Formulated and/or co-formulated liposome compositions containing toll-like receptor (“TLR”) agonist prodrugs useful in the treatment of cancer and methods thereof

ABSTRACT

Formulated and/or co-formulated liposomes comprising TLR prodrugs and/or TLR Lipid Moieties and methods of making the liposomes are disclosed herein. The TLR prodrug compositions comprise a drug moiety, a lipid moiety, and linkage unit that inhibit Toll-Like Receptor (e.g., TLR1/2, TLR4, and/or TLR7). The TLR prodrugs can be formulated and/or co-formulated into a liposome to provide a method of treating cancer, immunological disorders, and other disease by utilizing a targeted drug delivery vehicle.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/974,306, filed 21 Dec. 2020, which claims priority to U.S.Provisional Patent Application No. 62/974,746 filed 20 Dec. 2019, thecontents of which are fully incorporated by reference herein.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

FIELD OF THE INVENTION

The invention described herein relates to prodrug compositions thatinhibit toll-like receptor(s) (“TLR”) after release of the activeinhibitor from the prodrug and nano-formulations comprising suchprodrugs. Specifically, the invention relates to prodrug compositionswhich are formulated within a nanocarrier (e.g., a liposome) and used asa vehicle for cancer therapy in humans. The invention further relates tothe treatment of cancers and other immunological disorders and diseases.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of death next to coronary diseaseworldwide. Millions of people die from cancer every year and in theUnited States alone cancer kills well over a half-million peopleannually, with 1,688,780 new cancer cases diagnosed in 2017 (AmericanCancer Society). While deaths from heart disease have been decliningsignificantly, those resulting from cancer generally are on the rise. Inthe early part of the next century, cancer is predicted to become theleading cause of death unless medical developments change the currenttrend.

Several cancers stand out as having high rates of mortality. Inparticular, carcinomas of the lung (18.4% of all cancer deaths), breast(6.6% of all cancer deaths), colorectal (9.2% of all cancer deaths),liver (8.2% of all cancer deaths), and stomach (8.2% of all cancerdeaths) represent major causes of cancer death for both sexes in allages worldwide (GLOBOCAN 2018). These and virtually all other carcinomasshare a common lethal feature in that they metastasis to sites distantfrom the primary tumor and with very few exceptions, metastatic diseasefatal. Moreover, even for those cancer patients who initially survivetheir primary cancers, common experience has shown that their lives aredramatically altered. Many cancer patients experience strong anxietiesdriven by the awareness of the potential for recurrence or treatmentfailure. Many cancer patients also experience physical debilitationsfollowing treatment. Furthermore, many cancer patients experience arecurrence of their disease.

Although cancer therapy has improved over the past decades and survivalrates have increased, the heterogeneity of cancer still demands newtherapeutic strategies utilizing a plurality of treatment modalities.This is especially true in treating solid tumors at anatomical crucialsites (e.g., glioblastoma, squamous carcinoma of the head and neck andlung adenocarcinoma) which are sometimes limited to standardradiotherapy and/or chemotherapy. Nonetheless, detrimental effects ofthese therapies are chemo- and radio resistance, which promoteloco-regional recurrences, distant metastases and second primary tumors,in addition to severe side-effects that reduce the patients' quality oflife.

Toll-Like Receptors (“TLRs”) are a family of ten (10) identified patternrecognition receptors that respond to pathogen associated molecularpatterns (PAMPs) and self-derived damage-associated molecule patterns(DAMPs). The TLRs then activate downstream pathways that initiate aninnate immune response by producing inflammatory cytokines, type Iinterferon (IFN), and other mediators.

The TLR class of proteins are single, membrane-spanning, receptorsusually expressed on sentinel cells such as macrophages and dendriticcells that recognize conserved molecules derived from microbes. Oncethese microbes have breach physical barriers (e.g., skin, or intestinaltract mucosa) they are recognized by TLRs, which then activate immunecell responses.

Upon activation, TLRs recruit adapter proteins (i.e., proteins thatmediate other protein-protein interactions) within the cytosol of theimmune cell in order to propagate the antigen-induced signaltransduction pathway. These recruited proteins are then responsible forthe subsequent activation of other downstream proteins, includingprotein kinases (IKKi, IRAK1, IRAK4, and TBK1) that further amplify thesignal and ultimately lead to the upregulation or suppression of genesthat orchestrate inflammatory responses and other transcriptionalevents.

Within the TLR family, several members are worth noting. TLR1 and TLR2(“TLR 1/2”) are cell surface receptors that form heterodimers whichrecognize bacterial antigens such as lipoproteins as well as DAMPs suchas HMGB1, heat shock proteins, and proteoglycans. TLR1/2 are expressedin pre-dendritic cells, macrophage, and NK cells where they mediate theinnate response to PAMPS and DAMPS, upregulating inflammatory cytokinesand enhancing antigen processing. TLR1/2 agonists, such as PAM3CSK4, canalso enhance adaptive immunity as they have been shown to abrogate theimmune suppressive effects of Treg cells.

Additionally, TLR4 is a cell surface receptor for various bacterial andviral components, most notably Lipopolysaccharide (LPS). LPS, also knownas endotoxin, is a cell wall component of Gram-negative bacteria. LPShas been shown as a natural adjuvant for specific immune responses,especially antigen (Ag)-specific antibody and T cell responses. Thetoxicity associated with LPS has precluded its use as an effective andsafe vaccine adjuvant. However, the monophosphorylated lipid A (MPLA), ametabolic product of LPS, has been found to maintain many of theimmunostimulatory functions of LPS, but is significantly less toxic thanits parent. Accordingly, MPLA works well as a safe and effective vaccineadjuvant. Lipid A structural features known to account for themaintenance of adjuvant properties and the loss of toxicity include thenumber of phosphate groups, as well as the number, type, and location offatty acid residues. Synthetic MPLA, as well as a number of functionalanalogs, have been produced and characterized as immune stimulatingadjuvants. A number of these have been used clinically as adjuvants invaccine cocktails including in conjunction with tumor antigens toillicit anti-tumor immunity.

In addition, TLR4 is also a receptor for High Mobility Group Box 1(HMGB1), a protein secreted by tumor cells upon immunogenic cell-deaththat enhances anti-tumor immunity by recruitment of dendritic cells andstimulation of antigen processing and secretion of inflammatorycytokines by antigen presenting cells (APCs). Accordingly, co-deliveryof a TLR4 agonist with an ICD-inducing chemotherapy to a tumor enhancesthe anti-tumor immunity initiated by the ICD-chemotherapeutic.

Additionally, a prodrug is a medication or compound that, afteradministration, is metabolized (i.e., converted within the body) into apharmacologically active drug. Instead of administering a drug directly,a corresponding prodrug is used instead to improve how a medicine isabsorbed, distributed, metabolized, and/or excreted. Prodrugs are oftendesigned to improve bioavailability when a drug itself is poorlyabsorbed from the gastrointestinal tract, for example. A prodrug may beused to improve how selectively the drug interacts with cells orprocesses that are not its intended target. This reduces adverse orunintended effects of a drug, especially important in treatments likechemotherapy, which can have severe unintended and undesirable sideeffects. Prodrugs can thus be viewed as drugs containing specializednon-toxic protective groups used in a transient manner to alter or toeliminate undesirable properties in the parent molecule.

Finally, a nanocarrier is a nanomaterial being used as a transport foranother substance, such as a drug. There are many different types ofnanocarriers. For example, nanocarriers include polymer conjugates,polymeric nanoparticles, lipid-based carriers, and dendrimers to name afew. Different types of nanomaterial(s) being used in nanocarriersallows for hydrophobic and hydrophilic drugs to be delivered throughoutthe body. Since the human body contains mostly water, the ability todeliver hydrophobic drugs effectively in humans is a major therapeuticbenefit of nanocarriers. Nanocarriers show promise in the drug deliveryprocess because they can deliver drugs to site-specific targets,allowing drugs to be delivered in certain organs or cells but not inothers. Site-specificity is a major therapeutic benefit since itprevents drugs from being delivered to the wrong places. Additionally,nanocarriers show specific promise for use in chemotherapy because theycan help decrease the adverse, broader-scale toxicity of chemotherapy onhealthy, fast growing cells around the body. Since chemotherapy drugscan be extremely toxic to human cells, it is important that they aredelivered to the tumor without being released into other parts of thebody.

From the aforementioned, it will be readily apparent to those skilled inthe art that a new treatment paradigm is needed in the treatment ofcancers and other immunological diseases. By using novel prodrugs inconjunction with modern nanocarrier modalities, a new disease treatmentcan be achieved with the overall goal of more effective treatment(s),reduced side effects, and greater therapeutic utility in the treatmentof cancers, especially the treatment of cancers in solid tumors.

Given the current deficiencies associated with cancer treatment, it isan object of the present invention to provide new and improved methodsof treating cancer(s), immunological disorders, and other diseasesutilizing prodrugs encapsulated within a nanocarrier.

In the present disclosure, use of TLR agonists in conjunction withICD-inducing chemotherapeutics to illicit an immune response directlyagainst the actual patient's tumor cells in situ (i.e., without the needto introduce a tumor antigen or to remove tumor cells for ex vivotreatment). These synergistic functional agents are packaged into asingle nano-carrier vehicle ensuring co-delivery and enhanced tumorselectivity of the combination therapy.

SUMMARY OF THE INVENTION

The invention provides for TLR inhibitor prodrug (“TLR Prodrug”)compositions comprising a TLR inhibitor agent, a lipid, and abiologically cleavable linker. In certain embodiments, nanocarrierscomprising TLR Prodrug(s) are formulated for use as a delivery modalityto treat human diseases such as cancer, including solid tumor cancers aswell as other immunological disorders. In certain embodiments, thenanocarriers comprise a lipid-bilayer capable of being incorporated intoa drug delivery vehicle (i.e., a liposome). In a further preferredembodiment, the liposome comprises cholesterol hemisuccinate (“CHEMS”).In a further preferred embodiment, the liposome of the inventioncomprises Stearic Acid.

In a further embodiment, the invention comprises methods of delivering aTLR inhibitor to a tumor comprising (i) synthesizing a TLR prodrug; (ii)formulating a TLR prodrug of the invention in a nanocarrier of theinvention; and (iii) administering the nanocarrier to a patient.

In another embodiment, the invention comprises methods of delivering aTLR inhibitor with one or more additional immune modulating agent to atumor comprising (i) synthesizing a TLR prodrug; (ii) co-formulating aTLR prodrug of the invention in a nanocarrier with one or moreadditional immune modulating agents of the invention; and (iii)administering the nanocarrier to a patient.

In another embodiment, the immune modulating agents comprise agonists ofother TLRs, immunogenic-cell death (ICD) inducing chemotherapeutics,PD-1/PD-L1 antagonists, IDO antagonists, STING agonists, CTLA4inhibitors, iNKT cell agonists, and/or prodrugs thereof.

In another embodiment, the present disclosure teaches methods ofsynthesizing TLR prodrugs.

In another embodiment, the present disclosure teaches methods offormulating TLR prodrugs within nanocarriers, including but not limitedto liposomes.

In another embodiment, the present disclosure teaches methods oftreating cancer(s), immunological disorders and other diseases in humansusing nanocarriers of the present disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 . General Chemical Synthesis for TR5(B).

FIG. 2 . General Chemical Synthesis for TR6.

FIG. 3 . TLR Inhibitor Prodrug Synthesis Schema with Carboxylic AcidFunctionality.

FIG. 4 . TLR Inhibitor Prodrug Synthesis Schema with AlcoholFunctionality.

FIG. 5 . TLR Inhibitor Prodrug Synthesis Schema with Secondary Amine,Amide, or Aniline Functionality.

FIG. 6 . Chemical Synthesis for TR6 Prodrug Intermediate.

FIG. 7 . Chemical Synthesis for TR6 Prodrug Comprising CHEMS.

FIG. 8 . Chemical Synthesis for TR3.

FIG. 9 . Characterization of LNP-TR6 Liposome.

FIG. 10 . Characterization of LNP-TR6 Liposome (Zeta Potential).

FIG. 11 . Characterization of LNP-TR5 Liposome.

FIG. 12 . Characterization of LNP-TR5 Liposome (Zeta Potential).

FIG. 13 . Characterization of LNP-TR3 Liposome.

FIG. 14 . Characterization of LNP-TR3 Liposome (Zeta Potential).

FIG. 15 . Characterization of LNP-TR8 Liposome.

FIG. 16 . Characterization of LNP-TR8 Liposome (Zeta Potential).

FIG. 17 . Characterization of LNP-ID3-TR8 Liposome.

FIG. 18 . Characterization of LNP-ID3-TR8 Liposome (Zeta Potential).

FIG. 19 . Tumor Inhibition of LNP-TR5 in Combination with LNP-DOX inB16F10 Cancer Cells.

FIG. 20 . Tumor Inhibition of LNP-TR6 in Combination with LNP-NK1 andLNP-TR6 in Combination with LNP-TR8 and LNP-MTO in B16F10 Cancer Cells.

FIG. 21 . Tumor Inhibition of LNP-TR5 and LNP-TR6 in Combination withLNP-AR5 in B16F10 Cancer Cells.

FIG. 22 . Tumor Inhibition of LNP-TR5 in Combination with LNP-AR5 andLNP-DOX in EMT6 Cancer Cells.

FIG. 23 . Tumor Inhibition Studies of LNP-TR5 and LNP-TR6 in MultipleCombination(s) in H22 Cancer Cells.

FIG. 24 . Tumor Inhibition Studies of LNP-TR5 and LNP-TR6 in MultipleCombination(s) in Colorectal Cancer.

FIG. 25 . In Vitro Validation of TR6 Prodrug in Liposome Form Mechanismof Action.

FIG. 26 . In Vitro Validation of TR8 in Liposome Form Mechanism ofAction.

FIG. 27 . In Vitro Validation of TR5 in Liposome Form Mechanism ofAction.

FIG. 28 . In Vitro Validation of TR3 in Liposome Form Mechanism ofAction. 28(A). Raw-Blue™ Incubated with TR5 and LNP-TR5. 28(B).Raw-Blue™ Incubated with TR3, TR3+Stearic Acid, and LNP-TR3.

FIG. 29 . In Vitro Validation of TR3 in Liposome Form Mechanism ofAction. 29(A). HEK-Blue TLR8 Incubated with TR5 and LNP-TR5. 29(B).HEK-Blue TLR8 Incubated with TR3, TR3+Stearic Acid, and LNP-TR3Untreated Group.

DETAILED DESCRIPTION OF THE INVENTION Outline of Sections

I.) Definitions

II.) Prodrugs

III.) Drug Moieties

IV.) Lipids

V.) Linkage Unit(s) (“LU”)

VI.) Nanocarriers

VII.) Liposomes

VIII.) Pharmaceutical Formulation

IX.) Combination Therapy

X.) Methods of Delivering Liposomes Comprising Prodrugs to a Cell

XI.) Methods of Treating Cancer(s) and Other Immunological Disorder(s)

XII.) KITS/Articles of Manufacture

I.) Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains unless the context clearly indicates otherwise. Insome cases, terms with commonly understood meanings are defined hereinfor clarity and/or for ready reference, and the inclusion of suchdefinitions herein should not necessarily be construed to represent asubstantial difference over what is generally understood in the art.

When a trade name is used herein, reference to the trade name alsorefers to the product formulation, the generic drug, and the activepharmaceutical ingredient(s) of the trade name product, unless otherwiseindicated by context.

As used herein, the term “about”, when referring to a value or to anamount of size (i.e., diameter), weight, concentration or percentage ismeant to encompass variations of in one example ±20% or ±10%, in anotherexample ±5%, in another example ±1%, and in still another example ±0.1%from the specified amount, as such variations are appropriate to performthe disclosed methods.

As used herein, the term “and/or” when used in the context of a listingof entities, refers to the entities being present singly or incombination. Thus, for example, the phrase “A, B, C, and/or D” includesA, B, C, and D individually, but also includes any and all combinationsand sub combinations of A, B, C, and D.

Numerical ranges recited herein by endpoints include all numbers andfractions subsumed within that range (e.g., 1 to 5 includes, but is notlimited to, 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5).

As used herein, the phrase “consisting essentially of” limits the scopeof a claim to the specified materials or steps, plus those that do notmaterially affect the basic and novel characteristic(s) of the claimedsubject matter.

The terms “advanced cancer”, “locally advanced cancer”, “advanceddisease” and “locally advanced disease” mean cancers that have extendedthrough the relevant tissue capsule and are meant to include stage Cdisease under the American Urological Association (AUA) system, stageC1-C2 disease under the Whitmore-Jewett system, and stage T3-T4 and N+disease under the TNM (tumor, node, metastasis) system. In general,surgery is not recommended for patients with locally advanced diseaseand these patients have substantially less favorable outcomes comparedto patients having clinically localized (organ-confined) cancer.

As used herein the term “alkyl” can refer to C₁-C₂₀ inclusive, linear(i.e., “straight-chain”), branched, or cyclic, saturated, or at leastpartially and in some cases unsaturated (i.e., alkenyl and alkynyl)hydrocarbon chains, including for example, methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl,propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl,butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups. “Branched”refers to an alkyl group in which a lower alkyl group, such as methyl,ethyl, or propyl, is attached to a linear alkyl chain. “Lower alkyl”refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a C₁-C₈alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. “Higher alkyl”refers to an alkyl group having about 10 to about 20 carbon atoms, e.g.,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certainembodiments, “alkyl” refers, in particular, to C₁-C₈ straight-chainalkyls. In other embodiments, “alkyl” refers, in particular, toCi.₈branched-chain alkyls.

Alkyl groups can optionally be substituted (a “substituted alkyl”) withone or more alkyl group substituents, which can be the same ordifferent. The term “alkyl group substituent” includes but is notlimited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl,aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio,carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. In some embodiments,there can be optionally inserted along the alkyl chain one or moreoxygen, sulfur or substituted or unsubstituted nitrogen atoms, whereinthe nitrogen substituent is hydrogen, lower alkyl (also referred toherein as “alkylaminoalkyl”), or aryl.

Thus, as used herein, the term “substituted alkyl” includes alkylgroups, as defined herein, in which one or more atoms or functionalgroups of the alkyl group are replaced with another atom or functionalgroup, including for example, alkyl, substituted alkyl, halogen, aryl,substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino,dialkylamino, sulfate, and mercapto.

The term “aryl” is used herein to refer to an aromatic substituent thatcan be a single aromatic ring, or multiple aromatic rings that are fusedtogether, linked covalently, or linked to a common group, such as, butnot limited to, a methylene or ethylene moiety. The common linking groupalso can be a carbonyl, as in benzophenone, or oxygen, as indiphenylether, or nitrogen, as in diphenylamine. The term “aryl”specifically encompasses heterocyclic aromatic compounds. The aromaticring(s) can comprise phenyl, naphthyl, biphenyl, diphenylether,diphenylamine and benzophenone, among others. In particular embodiments,the term “aryl” means a cyclic aromatic comprising about 5 to about 10carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5-and 6-membered aromatic and heteroaromatic rings. The aryl group can beoptionally substituted (a “substituted aryl”) with one or more arylgroup substituents, which can be the same or different, wherein “arylgroup substituent” includes alkyl, substituted alkyl, aryl, substitutedaryl, aralkyl, hydroxyl, alkoxyl, aryloxyl, aralkyloxyl, carboxyl, acyl,halo, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl,acyloxyl, acylamino, aroylamino, carbamoyl, alkylcarbamoyl,dialkylcarbamoyl, arylthio, alkylthio, alkylene, and —NR′R″, wherein R′and R″ can each be independently hydrogen, alkyl, substituted alkyl,aryl, substituted aryl, and aralkyl. Specific examples of aryl groupsinclude, but are not limited to, cyclopentadienyl, phenyl, furan,thiophene, pyrrole, pyran, pyridine, imidazole, benzimidazole,isothiazole, isoxazole, pyrazole, pyrazine, triazine, pyrimidine,quinoline, isoquinoline, indole, carbazole, and the like.

“Heteroaryl” as used herein refers to an aryl group that contains one ormore non-carbon atoms (e.g., O, N, S, Se, etc.) in the backbone of aring structure. Nitrogen-containing heteroaryl moieties include, but arenot limited to, pyridine, imidazole, benzimidazole, pyrazole, pyrazine,triazine, pyrimidine, and the like.

The terms “anticancer drug”, “chemotherapeutic”, and “anticancerprodrug” refer to drugs (i.e., chemical compounds) or prodrugs known to,or suspected of being able to treat a cancer (i.e., to kill cancercells, prohibit proliferation of cancer cells, or treat a symptomrelated to cancer). In some embodiments, the term “chemotherapeutic” asused herein refers to a non-PS molecule that is used to treat cancerand/or that has cytotoxic ability. More traditional or conventionalchemotherapeutic agents can be described by mechanism of action or bychemical compound class, and can include, but are not limited to,alkylating agents (e.g., melphalan), anthracyclines (e.g., doxorubicin),cytoskeletal disruptors (e.g., paclitaxel), epothilones, histonedeacetylase inhibitors (e.g., vorinostat), inhibitors of topoisomerase Ior II (e.g., irinotecan or etoposide), kinase inhibitors (e.g.,bortezomib), nucleotide analogs or precursors thereof (e.g.,methotrexate), peptide antibiotics (e.g., bleomycin), platinum basedagents (e.g., cisplatin or oxaliplatin), retinoids (e.g., tretinoin),and vinka alkaloids (e.g., vinblastine).

“Aralkyl” refers to an -alkyl-aryl group, optionally wherein the alkyland/or aryl moiety is substituted.

“Alkylene” refers to a straight or branched bivalent aliphatichydrocarbon group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbonatoms. The alkylene group can be straight, branched, or cyclic. Thealkylene group also can be optionally unsaturated and/or substitutedwith one or more “alkyl group substituents.” There can be optionallyinserted along the alkylene group one or more oxygen, sulfur orsubstituted or unsubstituted nitrogen atoms (also referred to herein as“alkylaminoalkyi”), wherein the nitrogen substituent is alkyl aspreviously described. Exemplary alkylene groups include methylene(—CH₂—); ethylene (—CH₂—CH₂—); propylene (—(CH₂)3—); cyclohexylene(—C₆H₁₀—); —CH═CH—CH═CH—; —CH═CH—CH₂—; —(CH₂)_(q)—N(R)—(CH₂),—, whereineach of q is an integer from 0 to about 20, e.g., 0, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and R ishydrogen or lower alkyl; methylenedioxyl (-0-CH₂—0-); and ethylenedioxyl(-0-(CH₂)₂-0-). An alkylene group can have about 2 to about 3 carbonatoms and can further have 6-20 carbons.

The term “arylene” refers to a bivalent aromatic group, e.g., a bivalentphenyl or napthyl group. The arylene group can optionally be substitutedwith one or more aryl group substituents and/or include one or moreheteroatoms.

The term “amino” refers to the group —N(R)2 wherein each R isindependently H, alkyl, substituted alkyl, aryl, substituted aryl,aralkyl, or substituted aralkyl. The terms “aminoalkyl” and “alkylamino”can refer to the group —N(R)₂ wherein each R is H, alkyl or substitutedalkyl, and wherein at least one R is alkyl or substituted alkyl.“Arylamine” and “aminoaryl” refer to the group —N(R)₂ wherein each R isH, aryl, or substituted aryl, and wherein at least one R is aryl orsubstituted aryl, e.g., aniline (i.e., —NHC₆H₅).

A “bioreactive nanomaterial” refers to an engineered biomaterial thatinduces or catalyzes a biological response. In certain embodiments thenanomaterial induces a response by virtue of one or more propertiesselected from the group consisting of composition, size, shape, aspectratio, dissolution, electronic, redox, surface display, surface coating,hydrophobic, hydrophilic, an atomically thin nanosheet, orfunctionalized surface groups” to catalyze the biological response atvarious nano/bio interfaces. In certain embodiments the bioreactivenanomaterial has the ability to inhibit TLR-1 biological responses incells (e.g., in tumor cells) and/or as well as activating the innateimmune system through delivery of “danger signal” and adjuvant effects.

“Bulk” (a.k.a. Drug Substance) means the drug substance or the drugproduct which has not been filled into final containers fordistribution. Final formulated bulk generally refers to drug productwhich is formulated and being stored or held prior to filling. Drugsubstance may be stored or held as “bulk” or “concentrated bulk” priorto formulation into drug product.

The terms “carboxylate” and “carboxylic acid” can refer to the groups—C(═O)O— and —C(═O)OH, respectively. The term “carboxyl” can also referto the —C(═O)OH group.

The terms “conjugate” and “conjugated” as used herein can refer to theattachment (e.g., the covalent attachment) of two or more components(e.g., chemical compounds, polymers, biomolecule, particles, etc.) toone another. In some embodiments, a conjugate can comprise monovalentmoieties derived from two different chemical compounds covalently linkedvia a bivalent linker moiety (e.g., an optionally substituted alkyleneor arylene). In some embodiments, the linker can contain one or morebiodegradable bond, such that one or more bonds in the linker can bebroken when the prodrug is exposed to a particular physiologicalenvironment or enzyme (for example, esterases).

The term “compound” refers to and encompasses the chemical compound(e.g. a prodrug) itself as well as, whether explicitly stated or not,and unless the context makes clear that the following are to beexcluded: amorphous and crystalline forms of the compound, includingpolymorphic forms, where these forms may be part of a mixture or inisolation; free acid and free base forms of the compound, which aretypically the forms shown in the structures provided herein; isomers ofthe compound, which refers to optical isomers, and tautomeric isomers,where optical isomers include enantiomers and diastereomers, chiralisomers and non-chiral isomers, and the optical isomers include isolatedoptical isomers as well as mixtures of optical isomers including racemicand non-racemic mixtures; where an isomer may be in isolated form or ina mixture with one or more other isomers; isotopes of the compound,including deuterium- and tritium-containing compounds, and includingcompounds containing radioisotopes, including therapeutically- anddiagnostically-effective radioisotopes; multimeric forms of thecompound, including dimeric, trimeric, etc. forms; salts of thecompound, preferably pharmaceutically acceptable salts, including acidaddition salts and base addition salts, including salts having organiccounterions and inorganic counterions, and including zwitterionic forms,where if a compound is associated with two or more counterions, the twoor more counterions may be the same or different; and solvates of thecompound, including hemisolvates, monosolvates, disolvates, etc.,including organic solvates and inorganic solvates, said inorganicsolvates including hydrates; where if a compound is associated with twoor more solvent molecules, the two or more solvent molecules may be thesame or different. In some instances, reference made herein to acompound of the invention will include an explicit reference to one orof the above forms, e.g., salts and/or solvates; however, this referenceis for emphasis only, and is not to be construed as excluding other ofthe above forms as identified above.

“Drug product” means a final formulation that contains an active drugingredient (i.e., liposomes containing TLR inhibitor prodrugs)generally, but not necessarily, in association with inactiveingredients. The term also includes a finished dosage form that does notcontain an active ingredient but is intended to be used as a placebo.

The term “disulfide” can refer to the —S—S— group.

The term “empty vesicle” means an unloaded lipid vesicle by itself.

The term “ester” as used herein means a chemical compound derived fromacid (organic or inorganic) in which at least one —OH hydroxyl group isreplaced by an —O-alkyl (alkoxy) or O-Aryl (aryloxy) group.

The term “esterase” as used herein is a hydrolase enzyme that splitsesters into an acid and an alcohol.

“Excipient” means an inactive substance used as a carrier for the activeingredients in a drug such as vaccines. Excipients are also sometimesused to bulk up formulations with very potent active ingredients, toallow for convenient and accurate dosage. Examples of excipients includebut are not limited to, antiadherents, binders, coatings, disintegrants,fillers, dilutants, flavors, colors, lubricants, and preservatives.

The terms “halo”, “halide”, or “halogen” as used herein refer to fluoro,chloro, bromo, and iodo groups.

The terms “hydroxyl” and “hydroxy” refer to the —OH group.

The terms “inhibit” or “inhibition of” as used herein means to reduce bya measurable amount, or to prevent entirely.

The terms “individual” or “patient,” as used in the context of thisdisclosure can be used interchangeably.

As used herein, the term “ligand” refers generally to a species, such asa molecule or ion, which interacts, e.g., binds, in some way withanother species. See MARTELL, A. E., and HANCOCK, R. P., Metal Complexesin Aqueous Solutions, Plenum: New York (1996), which is incorporatedherein by reference in its entirety.

The term “lipid” as used herein refers to a class of naturally occurring(organic) compounds that are insoluble in polar solvents. In the contextof the disclosure, a lipid refers to conventional lipids, phospholipids,cholesterol, chemically functionalized lipids for attachment of PEG andligands, etc.

The term “lipid bilayer” or “LB” refers to any double layer of orientedamphipathic lipid molecules in which the hydrocarbon tails face inwardto form a continuous non-polar phase.

The term(s) “liposome” or “lipid vesicle” or “vesicle” are usedinterchangeably to refer to an aqueous compartment enclosed by a lipidbilayer, as being conventionally defined (see, STRYER (1981)Biochemistry, 2d Edition, W. H. Freeman & Co., p. 213).

The term “mammal” refers to any organism classified as a mammal,including mice, rats, rabbits, dogs, cats, cows, horses, and humans. Inone embodiment of the invention, the mammal is a mouse. In anotherembodiment of the invention, the mammal is a human.

The terms “mercapto” or “thiol” refer to the —SH group.

The terms “metastatic cancer” and “metastatic disease” mean cancers thathave spread to regional lymph nodes or to distant sites and are meant toinclude stage D disease under the AUA system and stage T×N×M+ under theTNM system.

The terms “nanocarrier”, “nanoparticle, and “nanoparticle drug carrier”are used interchangeably and refer to a nanostructure having an aqueous,solid, or polymeric interior core. In certain embodiments thenanocarrier comprises a lipid bilayer encasing (or surrounding orenveloping) the porous particle core. In certain embodiments thenanocarrier is a liposome, lipid nanoparticle (“LNP”) or a solid-lipidnanoparticle (“SLNP”).

The terms “nanoscale particle,” “nanomaterial,” “nanocarrier”, and“nanoparticle” refer to a structure having at least one region with adimension (e.g., length, width, diameter, etc.) of less than about 1,000nm. In some embodiments, the dimension is smaller (e.g., less than about500 nm, less than about 250 nm, less than about 200 nm, less than about150 nm, less than about 125 nm, less than about 100 nm, less than about80 nm, less than about 70 nm, less than about 60 nm, less than about 50nm, less than about 40 nm, less than about 30 nm or even less than about20 nm). In some embodiments, the dimension is between about 20 nm andabout 250 nm (e.g., about 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250nm).

The term “nanovesicle” refers to a “lipid vesicle” having a diameter (orpopulation of vesicles having a mean diameter) ranging from about 20 nm,or from about 30 nm, or from about 40 nm, or from about 50 nm up toabout 500 nm, or up to about 400 nm, or up to about 300 nm, or up toabout 200 nm, or up to about 150 nm, or up to about 100 nm, or up toabout 80 nm. In certain embodiments a nanovesicle has a diameter rangingfrom about 40 nm up to about 80 nm, or from about 50 nm up to about 70nm.

“Pharmaceutically acceptable” refers to a non-toxic, inert, and/orcomposition that is physiologically compatible with humans or othermammals.

“Pharmaceutical formulation” means the process in which differentchemical substances are combined to a pure drug substance to produce afinal drug product.

The term “phosphonate” refers to the —P(═O)(OR)₂ group, wherein each Rcan be independently H, alkyl, aralkyl, aryl, or a negative charge(i.e., wherein effectively there is no R group present to bond to theoxygen atom, resulting in the presence of an unshared pair of electronson the oxygen atom). Thus, stated another way, each R can be present orabsent, and when present is selected from H, alkyl, aralkyl, or aryl.

The term “phosphate” refers to the —OP(═O)(OR′)₂ group, where R′ is H ora negative charge.

The term “prodrug” means a medication or compound that, afteradministration, is metabolized into a pharmacologically active drug. Forthe purposes of this disclosure, a prodrug of the invention comprisesthree (3) components: (i) a drug moiety; (ii) a lipid moiety; and (iii)a linkage unit (“LU”).

The term “TLR prodrug” means a prodrug of the inventions wherein thedrug moiety comprises a TLR inhibitor.

The term “pyrolipid” refers to a conjugate of a lipid and a porphyrin,porphyrin derivative, or porphyrin analog. In some embodiments, thepyrolipid can comprise a lipid conjugate wherein a porphyrin or aderivative or analog thereof is covalently attached to a lipid sidechain. See, for example U.S. Patent Application Publication No.2014/0127763.

As used herein, the terms “specific”, “specifically binds” and “bindsspecifically” refer to the selective binding of nanocarrier of theinvention to the target TLR-1.

The term “supported lipid bilayer” means a lipid bilayer enclosing aporous particle core. This definition as set forth in the disclosure isdenoted because the lipid bilayer is located on the surface andsupported by a porous particle core. In certain embodiments, the lipidbilayer can have a thickness ranging from about 6 nm to about 7 nm whichincludes a 3-4 nm thickness of the hydrophobic core, plus the hydratedhydrophilic head group layers (each about 0.9 nm) plus two partiallyhydrated regions of about 0.3 nm each. In various embodiments, the lipidbilayer surrounding the liposome comprises a continuous bilayer orsubstantially continuous bilayer that effectively envelops and seals theTLR inhibitor.

The term “thioalkyl” can refer to the group —SR, wherein R is selectedfrom H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl,and substituted aryl. Similarly, the terms “thioaralkyl” and “thioaryl”refer to —SR groups wherein R is aralkyl and aryl, respectively.

As used herein “to treat” or “therapeutic” and grammatically relatedterms, refer to any improvement of any consequence of disease, such asprolonged survival, less morbidity, and/or a lessening of side effectswhich are the byproducts of an alternative therapeutic modality; as isreadily appreciated in the art, full eradication of disease is apreferred but albeit not a requirement for a treatment act.

The term “therapeutically effective amount” refers to the amount ofactive prodrug, nano-encapsulated prodrug, or pharmaceutical agent thatelicits the biological or medicinal response in a tissue, system,animal, individual or human.

The term “unsupported lipid bilayer” means an uncoated lipid bilayer ina lipid vesicle or liposome.

II.) Prodrugs

As shown in the present disclosure and for the purposes of thisinvention, a suitable prodrug is formed by conjugating a drug moiety ofthe invention (See, section entitled Drug Moieties) to a lipid moiety ofthe invention (See, section entitled Lipids) via an LU (See, sectionentitled Linkage Units) of the present disclosure. For the purposes ofthis disclosure, formation of a TLR prodrug can utilize severalstrategies. (See, for example, FIG. 3 , FIG. 4 , and FIG. 5 ).

Accordingly, in some embodiments, the prodrug is a drug-lipid moietycomprising a TLR inhibitor of the disclosure.

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the disclosure, wherein the TLR inhibitor inhibitsTLR1/2.

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the disclosure, wherein the TLR inhibitor inhibitsTLR4.

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the disclosure, wherein the TLR inhibitor inhibitsTLR8.

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the disclosure, wherein the TLR inhibitor inhibitsTLR7/8.

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the disclosure, wherein the TLR inhibitor inhibitsTLR1/2, and wherein the prodrug comprises a prodrug from Formula I.

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the disclosure, wherein the TLR inhibitor inhibitsTLR7.

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the disclosure, wherein the TLR inhibitor inhibitsTLR7, and wherein the prodrug comprises a prodrug from Formula II.

In one embodiment, the prodrug comprises the following chemicalstructure denoted Formula I:

Wherein, in exemplary embodiments of FORMULA I:

and

Y=H, OH;

Or

X=H; and

Y=CHEMS or Stearic Acid or CHEMS+Linker or Stearic Acid+Linker

In a further embodiment, the prodrug comprises the following chemicalstructure denoted Formula II:

Wherein, in exemplary embodiments of FORMULA II:

Y=CH₂, O, NH; and

Thus, in one embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of FORMULA I.

Thus, in one embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of FORMULA II.

In one embodiment, the prodrug is a drug-lipid moiety comprising a TLRinhibitor set forth in FIG. 1 .

In one embodiment, the prodrug is a drug-lipid moiety comprising a TLRinhibitor set forth in FIG. 2 .

In one embodiment, the prodrug is a drug-lipid moiety comprising a TLRinhibitor set forth in FIG. 7 .

In one embodiment, the prodrug is a drug-lipid moiety comprising a TLRinhibitor set forth in FIG. 8 .

In a further embodiment, the TLR prodrug is a drug-lipid moietycomprising a lipid of the disclosure.

In a further embodiment, the TLR prodrug is a drug-lipid moiety wherebythe lipid is CHEMS.

In a further embodiment, the TLR prodrug is a drug-lipid moiety wherebythe lipid is Stearic Acid.

In a further embodiment, the TLR prodrug is a drug-lipid moietycomprising a LU of the disclosure.

In a further embodiment, the TLR prodrug is a drug-lipid moiety wherebythe LU is a hydromethylcarbamate linker.

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the invention, wherein the TLR inhibitor comprises thechemical composition(s) TR6 and/or TR6(A).

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the invention, wherein the TLR inhibitor comprises TR6and/or TR6(A) and further comprises CHEMS.

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the invention, wherein the TLR inhibitor comprises TR6and/or TR6(A) and further comprises Stearic Acid.

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the invention, wherein the TLR inhibitor comprises thechemical composition(s) TR3, TR5, TR5(A) and/or TR5(B).

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the invention, wherein the TLR inhibitor comprises TR3,TR5, TR5(A) and/or TR5(B) and further comprises CHEMS.

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the invention, wherein the TLR inhibitor comprises TR3,TR5, TR5(A) and/or TR5(B) and further comprises Stearic Acid.

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the invention, wherein the TLR inhibitor comprises TR6and further comprises CHEMS and whereby the LU is a hydromethylcarbamatelinker.

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the invention, wherein the TLR inhibitor comprises TR6and further comprises Stearic Acid and whereby the LU is ahydromethylcarbamate linker.

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the inventions, wherein the TLR inhibitor comprises TR6and further comprises Stearic Acid having the following structure:

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor, wherein the TLR inhibitor inhibits TLR1/2, of theinvention(s), and wherein the TLR inhibitor comprises TR6 and furthercomprises Stearic Acid having the following structure:

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the invention, wherein the TLR inhibitor comprises TR5,TR5(A) and/or TR5(B) and further comprises CHEMS and whereby the LU is ahydromethylcarbamate linker.

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the invention, wherein the TLR inhibitor comprises TR5,TR5(A) and/or TR5(B) and further comprises Stearic Acid and whereby theLU is a hydromethylcarbamate linker.

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the inventions, wherein the TLR inhibitor comprisesTR5(B) and further comprises Stearic Acid having the followingstructure:

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor, wherein the TLR inhibitor inhibits TLR7, of theinvention(s), and wherein the TLR inhibitor comprises TR5(B) and furthercomprises Stearic Acid having the following structure:

In a further embodiment, the prodrug is a drug-lipid moiety comprisingan TLR inhibitor of the inventions, wherein the TLR inhibitor comprisesTR6 and further comprises CHEMS having the following structure:

In one embodiment, the prodrug is a drug-lipid moiety comprising a TLRinhibitor set forth in FIG. 7 .

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the invention, wherein the TLR inhibitor comprises thechemical composition(s) TR3.

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the invention, wherein the TLR inhibitor comprises TR3and further comprises CHEMS.

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the invention, wherein the TLR inhibitor comprises TR3and further comprises Stearic Acid.

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the invention, wherein the TLR inhibitor comprises TR3and further comprises CHEMS and whereby the LU is a hydromethylcarbamatelinker.

In a further embodiment, the prodrug is a drug-lipid moiety comprising aTLR inhibitor of the invention, wherein the TLR inhibitor comprises TR3and further comprises Stearic Acid and whereby the LU is ahydromethylcarbamate linker.

In a further embodiment, the prodrug is a drug-lipid moiety comprisingan TLR inhibitor of the inventions, wherein the TLR inhibitor comprisesTR3 having the following structure:

In additional embodiments of the disclosure the subject matter providesa TLR inhibitor prodrug comprising a lipid-conjugated therapeutic agentparent drug. In some embodiments, the prodrug comprises: (a) amonovalent drug moiety, (b) a monovalent lipid moiety, and (c) abivalent linker moiety comprising a linkage unit that will degrade invivo, such as a disulfide bond, wherein the monovalent drug moiety andthe monovalent lipid moiety are linked (e.g., covalently linked) throughthe linker. The monovalent drug moiety and the monovalent lipid moietiescan be monovalent derivatives of a chemical compound, and a lipid,respectively. For instance, the monovalent derivative can be adeprotonated derivative of a chemical compound or lipid that comprises ahydroxyl, thiol, amino, or carboxylic acid group.

In further embodiments of the disclosure the subject matter provides aTLR inhibitor prodrug comprising a lipid-conjugated therapeutic agentparent drug. In some embodiments, the prodrug comprises: (a) a bivalentdrug moiety, (b) a bivalent lipid moiety, and (c) a bivalent linkermoiety comprising a linkage that will degrade in vivo, wherein thebivalent drug moiety and the bivalent lipid moiety are linked (e.g.,covalently linked) through the linker. The bivalent drug moiety and thebivalent lipid moieties can be bivalent derivatives of a chemicalcompound and a lipid, respectively. For instance, the bivalentderivative can be a deprotonated derivative of a chemical compound orlipid that comprises a hydroxyl, thiol, amino, or carboxylic acid group.

One of ordinary skill in the art will appreciate and be enabled to makevariations and modifications to the disclosed embodiment withoutaltering the function and purpose of the invention disclosed herein.Such variations and modifications are intended within the scope of thepresent disclosure.

III.) Drug Moieties

Another aspect of the invention provides for novel TLR prodrugcompound(s) with the following formula(s) denoted TR3, TRS, TR5(A), TR6,and TR6(A).

One of skill in the art will appreciate that a compound is useful as aTLR inhibitor (e.g., inhibits TLR, for example TLR1/2, TLR4, TLR7, TLR8and/or TLR7/8). By way of brief background, TLR-1 (CD281) recognizespathogen-associated molecular pattern with a specificity forgram-positive bacteria. TLR-1 is found on the epithelial cell layer thatlines the small and large intestine and is an important player in themanagement of the gut microbiota and detection of pathogens. It is alsofound on the surface of macrophages and neutrophils. TLR1 recognizespeptidoglycan and (triacyl) lipopeptides in concert with TLR2 (as aheterodimer) and has been clearly shown to interact with TLR2. See,FARHAT, et. al., J. Leukoc. Biol. 83(3): 692-701 (2007) and JIN, et.al., Cell. 130(6): 1071-1082 (2007).

TLR2 (CD282) is a protein that in humans is encoded by the TLR2 gene.TLR2 is a membrane protein which is expressed on the surface of certaincells and recognizes foreign substances and passes on appropriatesignals to the cells of the immune system. TLR2 is expressed mostabundantly in peripheral blood leukocytes and mediates host response toGram-positive bacteria and yeast via stimulation of NF-κB. See,BARRELLO, et. al. Int. J. Immun. & Pharm: 24(3): 549-556 (2011). TLR2resides on the plasma membrane where it responds to lipid-containingPAMPs such as lipoteichoic acid and di- and tri-acylatedcysteine-containing lipopeptides. It does this by forming dimericcomplexes with either TLR 1 or TLR6 on the plasma membrane. See, BOTOS,et. al., Structure 19(4): 447-459 (2011).

TLR4 (CD284) is another member of the TLR family. Its activation leadsto an intracellular signaling pathway NF-κB and inflammatory cytokineproduction which is responsible for activating the innate immune system.It is most well-known for recognizing lipopolysaccharide (LPS), acomponent present in many Gram-negative bacteria (e.g., Neisseria spp.)and select Gram-positive bacteria. Its ligands also include severalviral proteins, polysaccharide, and a variety of endogenous proteinssuch as low-density lipoprotein, beta-defensins, and heat shock protein.See, BRUBAKER, et. al., Annual Rev. of Immun. 33:257-290 (2015). TLR4signaling responds to signals by forming a complex using anextracellular leucine-rich repeat domain (LRR) and an intracellulartoll/interleukin-1 receptor (TIR) domain. LPS stimulation induces aseries of interactions with several accessory proteins which form theTLR4 complex on the cell surface. LPS recognition is initiated by an LPSbinding to an LBP protein. The conformational changes of the TLR4 inducethe recruitment of intracellular adaptor proteins containing the TIRdomain which is necessary to activate the downstream signaling pathway.LU, et. al., Cytokine 42(2): 145-151 (2008). TLR4 is capable ofactivating MAPK and NF-κB pathways, implicating possible direct role ofcell-autonomous TLR4 signaling in regulation of carcinogenesis, inparticular, through increased proliferation of tumor cells, apoptosisinhibition and metastasis.

TLR7 is another member of the TLR family. TLR7 recognizessingle-stranded RNA in endosomes, which is a common feature of viralgenomes which are internalized by macrophages and dendritic cells. TLR7recognizes single-stranded RNA of viruses such as HIV and HCV. See,HEIL, et. al., Science 303(5663): 1526-1529 (2004). TLR7 can recognizeGU-rich single-stranded RNA. Id. However, the presence of GU-richsequences in the single-stranded RNA is not sufficient to stimulateTLR7. TLR7 has been shown to play a significant role in the pathogenesisof autoimmune disorders such as Systemic Lupus Erythematosus (SLE) aswell as in the regulation of antiviral immunity. In addition, due totheir ability to induce robust production of anti-cancer cytokines suchas interleukin-12, TLR7 agonists have been investigated for cancerimmunotherapy.

Based on the foregoing, the present disclosure describes a class of TLRinhibitors.

In one embodiment, the class of TLR inhibitors inhibit TLR1/2.

In one embodiment, the class of TLR inhibitors inhibit TLR7.

In one embodiment, a drug moiety of the disclosure comprises a compoundwith the following chemical structure (denoted TR6):

In an alternative embodiment, a drug moiety of the disclosure comprisesa compound with the following chemical structure (denoted TR6(A)):

In one embodiment, a drug moiety of the disclosure comprises a compoundwith the following chemical structure (denoted TR5):

In an alternative embodiment, a drug moiety of the disclosure comprisesa compound with the following chemical structure (denoted TR5(A)):

In one embodiment, a drug moiety of the disclosure comprises a compoundwith the following chemical structure(s) (denoted TR3):

Wherein Y=CH₃, n-Pr, OEt, or NHEt; andWherein R=Saturated C12-C24 alkyl.

One of ordinary skill in the art will appreciate and be enabled to makevariations and modifications to the disclosed embodiment withoutaltering the function and purpose of the invention disclosed herein.Such variations and modifications are intended within the scope of thepresent disclosure.

IV.) Lipids

Generally speaking, and for the purposes of this disclosure, the term“lipid” is used in its broadest sense and comprises severalsub-categories of lipids, including but not limited to,phospholipids/fatty acids. As it is appreciated by one of skill in theart, a phospholipid represents a class of lipids that are a majorcomponent of all cell membranes. Phospholipids can form lipid bilayersbecause of their amphiphilic characteristic. The structure of thephospholipid molecule generally consists of two hydrophobic fatty acid“tails” and a hydrophilic “head” consisting of a phosphate group thatcan be modified with simple organic molecules such as choline,ethanolamine, or serine. These two components are usually joinedtogether by a glycerol molecule. A representative list ofphospholipids/fatty acid(s) of the invention are set forth in Table III.

By way of brief background, at the most fundamental level, theproperties of a liposome depend upon the subtle physicochemicalinteractions among the various lipid species in its composition.Individual lipids can be combined to form a myriad of superstructuresincluding bilayers, and bilayer properties can be tuned to modulate drugrelease and membrane stability. In a simplified bilayer model acyl chainlength dictates bilayer thickness and phase transition temperature (Tm),acyl chain saturation controls bilayer fluidity, and headgroupinteractions impact inter- and intra-lipid molecular forces. Liposomebehavior can be adjusted by incorporating synthetic lipids such as lipidprodrugs, fusogenic lipids and functionalizable lipids into the bilayer.See, KOHLI, et. al., J. Control Release, 0: pp. 274-287 (Sep. 28, 2014).

In one embodiment of the present disclosure, a TLR prodrug comprises amonovalent lipid moiety.

In one embodiment, a TLR prodrug comprises a bivalent lipid moiety.

In one embodiment, the lipid comprises a cholesterol with the followingchemical structure:

In one embodiment, the lipid comprises a DPPG with the followingchemical structure:

In one embodiment, the lipid comprises a DMPG with the followingchemical structure:

In one embodiment, the lipid comprises a Lyso PC with the followingchemical structure:

In one embodiment, the lipid comprises a (Δ9-Cis) PG.

In one embodiment, the lipid comprises a Soy Lyso PC with the followingchemical structure:

In one embodiment, the lipid comprises a PG with the following chemicalstructure:

In one embodiment, the lipid comprises a C16 PEG2000 Ceramde with thefollowing chemical structure:

In one embodiment, the lipid comprises a cholesterol hemisuccinate(“CHEMS”) with the following chemical structure:

In one embodiment, the lipid comprises a class of lipids having thefollowing chemical structure denoted Formula III:

Wherein, in exemplary embodiments of FORMULA III:

In one embodiment, a lipid moiety of the disclosure comprises a compoundwith the following chemical structure (denoted TR8):

See, GIGG J., et. al., Carb. Res. 141(1): pp. 91-97 (1985).

In one embodiment, the lipid comprises a class of lipids having thefollowing chemical structure denoted Formula IV:

Wherein, in exemplary embodiments of FORMULA IV:

In one embodiment, a lipid moiety of the disclosure comprises a compoundwith the following chemical structure (denoted TR11):

See, KAUR, et. al., RSC Advances 8, pp. 9587-9686 (2018).

In a further embodiment, a lipid moiety of the disclosure comprises aclass of invariant natural killer T (iNKT) cells.

In a further embodiment, a lipid moiety of the disclosure comprisesAlpha-galactosylceramide (α-GalCer).

By way of reference, a further list of the chemical formulas andabbreviation(s) of the lipids disclosed herein is set forth in Table I.

In an additional embodiment, the lipid comprises a phospholipid/fattyacid disclosed herein and set forth in Table Ill.

In a further embodiment, the lipid comprises a Stearic acid.

In addition, the TLR prodrugs and/or liposome(s) of the disclosure maycomprise one or more helper lipids which are also referred to herein as“helper lipid components”. The helper lipid components are preferablyselected from the group comprising phospholipids and steroids.Phospholipids are preferably di- and monoester of the phosphoric acid.Preferred members of the phospholipids are phosphoglycerides andsphingolipids. Steroids, as used herein, are naturally occurring andsynthetic compounds based on the partially hydrogenatedcyclopenta[a]phenanthrene. Preferably, the steroids contain 21 to 30 Catoms. A particularly preferred steroid is cholesterol.

It is to be noted that although not wishing to be bound by any theory,due to the particular mol percentages of the helper lipid(s) containedin the lipid compositions according to the present invention, whichhelper lipid can be either a PEG-free helper lipid or in particular aPEG-containing helper lipid, surprising effects can be realized, moreparticularly if the content of any of this kind of helper lipid iscontained within the concentration range specified herein.

In a further aspect of the present invention, lipid compositions whichare preferably present as lipoplexes or liposomes, preferably show aneutral or overall anionic charge. The anionic lipid is preferably anyneutral or anionic lipid described herein. The lipid compositioncomprises in a preferred embodiment any helper lipid or helper lipidcombination as well as any TLR inhibitor as described herein. In afurther embodiment the composition according to the present inventioncontaining nucleic acid(s) forms lipoplexes. In a preferred embodimentthe term lipoplexes as used herein refers to a composition composed ofneutral or anionic lipid, neutral helper lipid and TLR inhibitor of theinvention. For reference into the usage of helper lipids in the art,see, by way of example, U.S. Patent Application Publication2011/0178164; OJEDA, et. al., Int. J. of Pharmaceutics (March 2016);DABKOWSKA, et. al., J. R. Soc. Interface 9, pp. 548-561 (2012); andMOCHIZUKI, et. al., Biochimica et. Biophysica Acta, 1828, pp. 412-418(2013).

In a preferred embodiment, the helper lipids of the invention comprisethe helper lipids set forth in Table II.

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is CHEMS and wherein the drug moiety is TR6.

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is CHEMS and wherein the drug moiety is TR6, furthercomprising a LU and wherein the LU is a hydromethylcarbamate linker.

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is CHEMS and wherein the drug moiety is TR6, furthercomprising a LU and wherein the LU is a hydromethylcarbamate linker,further comprising a helper lipid component, wherein the helper lipidcomponent comprises a helper lipid of Table II.

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is CHEMS and wherein the drug moiety is TR6 andwherein the CHEMS is monovalent.

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is Stearic Acid and wherein the drug moiety is TR6.

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is Stearic Acid and wherein the drug moiety is TR6 andwherein the Stearic Acid is monovalent.

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is Stearic Acid and wherein the drug moiety is TR6,further comprising a LU and wherein the LU is a hydromethylcarbamatelinker.

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is Stearic Acid and wherein the chemical compositionis TR6, further comprising a LU and wherein the LU is ahydromethylcarbamate linker, further comprising a helper lipidcomponent, wherein the helper lipid component comprises a helper lipidof Table II.

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is CHEMS and wherein the drug moiety is TR5(B).

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is CHEMS and wherein the drug moiety is TR5(B),further comprising a LU and wherein the LU is a hydromethylcarbamatelinker.

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is CHEMS and wherein the drug moiety is TR5(B),further comprising a LU and wherein the LU is a hydromethylcarbamatelinker, further comprising a helper lipid component, wherein the helperlipid component comprises a helper lipid of Table II.

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is CHEMS and wherein the drug moiety is TR5(B) andwherein the CHEMS is monovalent.

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is Stearic Acid and wherein the drug moiety is TR5(B).

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is Stearic Acid and wherein the drug moiety is TR5(B)and wherein the Stearic Acid is monovalent.

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is Stearic Acid and wherein the drug moiety is TR5(B),further comprising a LU and wherein the LU is a hydromethylcarbamatelinker.

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is Stearic Acid and wherein the chemical compositionis TR5(B), further comprising a LU and wherein the LU is ahydromethylcarbamate linker, further comprising a helper lipidcomponent, wherein the helper lipid component comprises a helper lipidof Table II.

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is CHEMS and wherein the drug moiety is TR3.

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is CHEMS and wherein the drug moiety is TR3, furthercomprising a LU and wherein the LU is a hydromethylcarbamate linker.

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is CHEMS and wherein the drug moiety is TR3, furthercomprising a LU and wherein the LU is a hydromethylcarbamate linker,further comprising a helper lipid component, wherein the helper lipidcomponent comprises a helper lipid of Table II.

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is CHEMS and wherein the drug moiety is TR3 andwherein the CHEMS is monovalent.

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is Stearic Acid and wherein the drug moiety is TR3.

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is Stearic Acid and wherein the drug moiety is TR3 andwherein the Stearic Acid is monovalent.

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is Stearic Acid and wherein the drug moiety is TR3,further comprising a LU and wherein the LU is a hydromethylcarbamatelinker.

In one embodiment, a TLR prodrug comprises a lipid of the invention,wherein the lipid is Stearic Acid and wherein the chemical compositionis TR3, further comprising a LU and wherein the LU is ahydromethylcarbamate linker, further comprising a helper lipidcomponent, wherein the helper lipid component comprises a helper lipidof Table II.

One of ordinary skill in the art will appreciate and be enabled to makevariations and modifications to the disclosed embodiment withoutaltering the function and purpose of the invention disclosed herein.Such variations and modifications are intended within the scope of thepresent disclosure.

V.) Linkage Unit(s) (“LU”)

In some embodiments, the presently disclosed subject matter providesprodrugs comprising drug-lipid conjugates that include biodegradablelinkages, such as esters, thioesters, and other linkers known in theart.

Exemplary embodiments of ester chemistry are set forth herein:

In some embodiments, the prodrug is a drug-lipid conjugate, whereby thedrug-lipid conjugate is cleaved by an esterase.

In one embodiment, a prodrug of the invention comprises a LU via asecondary amine, amide, or aniline using the following schema:

An exemplary synthesis is as follows:

Cleavage of the prodrug structure comprising a secondary amine, amide,or aniline is obtained via esterase hydrolysis of the secondary amine,amide, or aniline prodrug under the following exemplary synthesis:

Esterase Hydrolysis of Secondary Amino/Amide/Aniline Prodrug

Wherein:R₁ and R₂ can be and molecule which connects a N via a C.

In one embodiment, the secondary amide nitrogen of the TR6 drug moietyis conjugated to CHEMS via a hydromethylcarbamate linker.

In one embodiment, the secondary amide nitrogen of the TR6(A) drugmoiety is conjugated to CHEMS via a hydromethylcarbamate linker.

In one embodiment, the secondary amide nitrogen of the TR5 drug moietyis conjugated to CHEMS via a hydromethylcarbamate linker.

In one embodiment, the secondary amide nitrogen of the TR5(A) drugmoiety is conjugated to CHEMS via a hydromethylcarbamate linker.

In one embodiment, the secondary amide nitrogen of the TR5(B) drugmoiety is conjugated to CHEMS via a hydromethylcarbamate linker.

One of ordinary skill in the art will appreciate and be enabled to makevariations and modifications to the disclosed embodiment withoutaltering the function and purpose of the invention disclosed herein.Such variations and modifications are intended within the scope of thepresent disclosure.

VI.) Nanocarrier(s)

Generally speaking, and for the purposes of this disclosurenanocarrier(s) are within the scope of the invention. A nanocarrier isnanomaterial being used as a transport module for another substance,such as a drug. Commonly used nanocarriers include micelles, polymers,carbon-based materials, liposomes, and other substances. Because oftheir small size, nanocarriers can deliver drugs to otherwiseinaccessible sites around the body. Nanocarriers can include polymerconjugates, polymeric nanoparticles, lipid-based carriers, dendrimers,carbon nanotubes, and gold nanoparticles. Lipid-based carriers includeboth liposomes and micelles.

In addition, nanocarriers are useful in the drug delivery processbecause they can deliver drugs to site-specific targets, allowing drugsto be delivered in certain organs or cells but not in others.Site-specificity poses a major therapeutic benefit since it preventsdrugs from being delivered to the wrong places. In addition,nanocarriers show promise for use in chemotherapy because they can helpdecrease the adverse, broader-scale toxicity of chemotherapy on healthy,fast growing cells around the body. Since chemotherapy drugs can beextremely toxic to human cells, it is important that they are deliveredto the tumor without being released into other parts of the body.

Generally speaking, there are four (4) methods in which nanocarriers candeliver drugs and they include passive targeting, active targeting, pHspecificity, and temperature specificity.

Passive targeting refers to a nanocarrier's ability to travel down atumor's vascular system, become trapped, and accumulate in the tumor.This accumulation is caused by the enhanced permeability and retentioneffect. The leaky vasculature of a tumor is the network of blood vesselsthat form in a tumor, which contain many small pores. These pores allownanocarriers in, but also contain many bends that allow the nanocarriersto become trapped. As more nanocarriers become trapped, the drugaccumulates at the tumor site. This accumulation causes large doses ofthe drug to be delivered directly to the tumor site.

Active targeting involves the incorporation of targeting modules such asligands or antibodies on the surface of nanocarriers that are specificto certain types of cells around the body. Generally, nanocarriers havea high surface-area to volume ratio allowing for multiple ligands to beincorporated on their surfaces.

Additionally, certain nanocarriers will only release the drugs theycontain in specific pH ranges. pH specificity also allows nanocarriersto deliver drugs directly to a tumor site. This is due to the fact thattumors are generally more acidic than normal human cells, with a pHaround 6.8. Normal tissue has a pH of around 7.4. Thus, nanocarriersthat only release drugs at certain pH ranges can therefore be used torelease the drug only within acidic tumor environments. High acidicenvironments cause the drug to be released due to the acidic environmentdegrading the structure of the nanocarrier. Generally, thesenanocarriers will not release drugs in neutral or basic environments,effectively targeting the acidic environments of tumors while leavingnormal body cells untouched. This pH sensitivity can also be induced inmicelle systems by adding copolymer chains to micelles that have beendetermined to act in a pH independent manor. See, WU, et. al.,Biomaterials, 34(4): 1213-1222 (2012). These micelle-polymer complexesalso help to prevent cancer cells from developing multi-drug resistance.The low pH environment triggers a quick release of the micelle polymers,causing a majority of the drug to be released at once, rather thangradually like other drug treatments.

Additionally, some nanocarriers have also been shown to deliver drugsmore effectively at certain temperatures. Since tumor temperatures aregenerally higher than temperatures throughout the rest of the body,around 40° C., this temperature gradient helps act as safeguard fortumor-specific site delivery. See, REZAEI, et. al., Polymer, 53(16):3485-3497 (2012).

As disclosed herein, lipid-based nanocarriers, such as liposomes arewithin the scope of this invention. Lipid-based nanoparticles (LBNPs orLNPs) such as liposomes, solid lipid nanoparticles (SLN) andnanostructured lipid carriers (NLC) can transport hydrophobic andhydrophilic molecules, display exceptionally low or no toxicity, andincrease the time of drug action by means of a prolonged half-life and acontrolled release of the drug. Lipid nanoparticles can include chemicalmodifications to avoid the detection by the immune system (gangliosidesor polyethylene glycol (PEG)) or to improve the solubility of the drug.In addition, they can be prepared in formulations sensitive to the pH inorder to promote drug release in an acid environment and can also beassociated with small molecules or antibodies that recognize tumor cellsor their receptors (such as folic acid (FoA)). Nanodrugs can also beused in combination with other therapeutic strategies to improve theresponse of patients. See, GARCIA-PINEL, et. al., Nanomaterials 9(639)(2019).

In various embodiments silicasome drug carriers described hereincomprise a porous silica (or other material) nanoparticle (e.g., asilica body having a surface and defining a plurality of pores that aresuitable to receive molecules therein) coated with a lipid bilayer. Thefact that the nanoparticle is referred to as a silica nanoparticle doesnot preclude materials other than silica from also being incorporatedwithin the silica nanoparticle. In some embodiments, the silicananoparticle may be substantially spherical with a plurality of poreopenings through the surface providing access to the pores. However, invarious embodiments the silica nanoparticle can have shapes other thansubstantially spherical shapes. Thus, for example, in certainembodiments the silica nanoparticle can be substantially ovoid,rod-shaped, a substantially regular polygon, an irregular polygon, andthe like.

Generally, the silica nanoparticle comprises a silica body that definesan outer surface between the pore openings, as well as side walls withinthe pores. The pores can extend through the silica body to another poreopening, or a pore can extend only partially through the silica bodysuch that that it has a bottom surface of defined by the silica body.

In some embodiments, the silica body is mesoporous. In otherembodiments, the silica body is microporous. As used herein,“mesoporous” means having pores with a diameter between about 2 nm andabout 50 nm, while “microporous” means having pores with a diametersmaller than about 2 nm. In general, the pores may be of any size, butin typical embodiments are large enough to contain one or moretherapeutic compounds therein. In such embodiments, the pores allowsmall molecules, for example, therapeutic compounds such as anticancercompounds to adhere or bind to the inside surface of the pores, and tobe released from the silica body when used for therapeutic purposes. Insome embodiments, the pores are substantially cylindrical.

In certain embodiments the nanoparticles comprise pores having porediameters between about 1 nm and about 10 nm in diameter or betweenabout 2 nm and about 8 nm. In certain embodiments the nanoparticlescomprise pores having pore diameters between about 1 nm and about 6 nm,or between about 2 nm and about 5 nm. Other embodiments includeparticles having pore diameters less than 2.5 nm.

In other embodiments, the pore diameters are between 1.5 and 2.5 nm.Silica nanoparticles having other pore sizes may be prepared, forexample, by using different surfactants or swelling agents during thepreparation of the silica nanoparticles. In various embodiments thenanoparticles can include particles as large (e.g., average or mediandiameter (or another characteristic dimension) as about 1000 nm.However, in various embodiments the nanoparticles are typically lessthan 500 nm or less than about 300 nm as, in general, particles largerthan 300 nm may be less effective in entering living cells or bloodvessel fenestrations. In certain embodiments the nanoparticles range insize from about 40 nm, or from about 50 nm, or from about 60 nm up toabout 100 nm, or up to about 90 nm, or up to about 80 nm, or up to about70 nm. In certain embodiments the nanoparticles range in size from about60 nm to about 70 nm. Some embodiments include nanoparticles having anaverage maximum dimension between about 50 nm and about 1000 nm. Otherembodiments include nanoparticles having an average maximum dimensionbetween about 50 nm and about 500 nm. Other embodiments includenanoparticles having an average maximum dimension between about 50 nmand about 200 nm.

In some embodiments, the average maximum dimension is greater than about20 nm, greater than about 30 nm, greater than 40 nm, or greater thanabout 50 nm. Other embodiments include nanoparticles having an averagemaximum dimension less than about 500 nm, less than about 300 nm, lessthan about 200 nm, less than about 100 nm or less than about 75 nm. Asused herein, the size of the nanoparticle refers to the average ormedian size of the primary particles, as measured by transmissionelectron microscopy (TEM) or similar visualization techniques known inthe art. Further examples of mesoporous silica nanoparticles include,but are not limited to, MCM-41, MCM-48, and SBA-15. See, KATIYARE, et.al., J. Chromotog. 1122(1-2): 13-20 (2006).

Methods of making porous silica nanoparticles are well known to those ofskill in the art. In certain embodiments mesoporous silica nanoparticleare synthesized by reacting tetraethyl orthosilicate (TEOS) with atemplate made of micellar rods. The result is a collection of nano-sizedspheres or rods that are filled with a regular arrangement of pores. Thetemplate can then be removed by washing with a solvent adjusted to theproper pH (See, e.g., TREWYN et al. (2007) Chem. Eng. J. 137(1): 23-29).

In certain embodiments mesoporous particles can also be synthesizedusing a simple sol-gel method (See, e.g., NANDIYANTO, et al. (2009)Microporous and Mesoporous Mat. 120(3): 447-453). In certain embodimentstetraethyl orthosilicate can also be used with an additional polymermonomer as a template. In certain embodiments3-mercaptopropyl)trimethoxysilane (MPTMS) is used instead of TEOS.

In certain embodiments the mesoporous silica nanoparticles are cores aresynthesized by a modification of the sol/gel procedure described by MENGet. al. (2015) ACS Nemo, 9(4): 3540-3557.

While the methods described herein have been demonstrated with respectto porous silica nanoparticles (e.g., mesoporous silica), it will berecognized by those skilled in the art that similar methods can be usedwith other porous nanoparticles. Numerous other mesoporous materialsthat can be used in drug delivery nanoparticles are known to those ofskill in the art. For example, in certain embodiments mesoporous carbonnanoparticles could be utilized.

Mesoporous carbon nanoparticles are well known to those of skill in theart (See, e.g., HUANG et. al. (2016) Carbon, 101: 135-142; ZHU et. al.(2014) Asian J. Pharm. Sci., 9(2): 82-91; and the like).

Similarly, in certain embodiments, mesoporous polymeric particles can beutilized. The syntheses of highly ordered mesoporous polymers and carbonframeworks from organic-organic assembly of triblock copolymers withsoluble, low-molecular-weight phenolic resin precursors (resols) by anevaporation induced self-assembly strategy have been reported by MENG,et. al. (2006) Chem. Mat. 6(18): 4447-4464.

The nanoparticles described herein are illustrative and non-limiting.Using the teachings provided herein numerous other lipid bilayer coatednanoparticles will be available to one of skill in the art.

In one embodiment, the invention teaches nanocarriers which comprise TLRprodrugs.

In one embodiment, the invention teaches nanocarriers which comprise TLRprodrugs, wherein the TLR prodrug comprises TR3.

In one embodiment, the invention teaches nanocarriers which comprise TLRprodrugs, wherein the TLR prodrug comprises TR6.

In one embodiment, the invention teaches nanocarriers which comprise TLRprodrugs, wherein the TLR prodrug comprises TR5.

In one embodiment, the invention teaches a nanocarrier comprising aliposome, wherein the lipid comprises CHEMS.

In one embodiment, the invention teaches a nanocarrier comprising aliposome, wherein the lipid comprises Stearic Acid.

In one embodiment, the invention teaches a nanocarrier comprising aliposome, wherein the lipid comprises CHEMS and whereby the liposomefurther comprises a TLR prodrug.

In one embodiment, the invention teaches a nanocarrier comprising aliposome, wherein the lipid comprises CHEMS and whereby the liposomefurther comprises TR6.

In one embodiment, the invention teaches a nanocarrier comprising aliposome, wherein the lipid comprises CHEMS and whereby the liposomefurther comprises TR3.

In one embodiment, the invention teaches a nanocarrier comprising aliposome, wherein the lipid comprises CHEMS and whereby the liposomefurther comprises TR5.

In one embodiment, the invention teaches a nanocarrier comprising aliposome, wherein the lipid comprises CHEMS and whereby the liposomefurther comprises TR5(B).

In one embodiment, the invention teaches a nanocarrier comprising aliposome, wherein the lipid comprises Stearic Acid and whereby theliposome further comprises a TLR inhibitor.

In one embodiment, the invention teaches a nanocarrier comprising aliposome, wherein the lipid comprises Stearic Acid and whereby theliposome further comprises TR3.

In one embodiment, the invention teaches a nanocarrier comprising aliposome, wherein the lipid comprises Stearic Acid and whereby theliposome further comprises TR6.

In one embodiment, the invention teaches a nanocarrier comprising aliposome, wherein the lipid comprises Stearic Acid and whereby theliposome further comprises TR5(B).

In one embodiment, the invention teaches nanocarriers which comprise TLRlipid moieties.

In one embodiment, the invention teaches a nanocarrier comprising a TLRlipid moiety, whereby the lipid moiety further comprises TR8.

In one embodiment, the invention teaches a nanocarrier comprising a TLRlipid moiety, whereby the lipid moiety further comprises TR11.

In one embodiment, the invention teaches a nanocarrier comprising a TLRlipid moiety, whereby the lipid moiety further comprisesAlpha-galactosylceramide (α-GalCer).

The scope of the disclosure teaches three (3) non-limiting possibletreatment modalities using the formulated prodrugs of the invention.See, PCT Patent Publication No. WO2018/213631.

The first treatment modality involves combination of a TLR prodrug incombination with another therapeutic (e.g., another formulated prodrugwhich inhibits TLR (e.g., TLR1/2, TLR4, TLR7, TLR8, and/or TLR7/8), achemotherapy agent (such as an ICD-inducing chemotherapy), etc.) into asingle liposome that allows systemic (or local) biodistribution and drugdelivery to tumor sites. The dual-delivery approach achieved synergisticenhancement of adaptive and innate immunity, leading to a significantimprovement in animal survival. In certain embodiments the nanocarriercomprises a vesicle (i.e., a lipid bilayer enclosing a fluid).

A second treatment modality involves local delivery to a tumor orperi-tumoral region, of an agent that inhibits TLR in combination with alipid (e.g., a liposome) that comprises an inhibitor of TLR (e.g.,TLR1/2, TLR4, TLR7, TLR8, and/or TLR7/8). It is demonstrated that suchlocal delivery of a TLR inhibitor in combination with a TLR prodruginduces cytotoxic tumor killing, and tumor shrinkage at the local site.These adaptive immune responses are accompanied by boosting of theinnate immune system, as reflected by CRT expression, as well as theactivation of a DC population, particularly well-suited for generatingcytotoxic T cell responses.

A third treatment modality involves vaccination utilizing dying cancercells {e.g., KPC cells) in which inhibition of TLR is induced ex vivo.It is discovered that such vaccination can generate a systemic immuneresponse that can interfere with tumor growth at a remote site as wellas allowing adoptive transfer to non-immune animals. One of skill in theart will appreciate and be enabled to perform methods the treatmentmodalities provided herein.

VII.) Liposomes

In one aspect, the presently disclosed subject matter is based on anapproach for providing a prodrug of the disclosure (See, sectionentitled Prodrugs) suitable for incorporation into a nanocarriercomprising lipid coating layers to provide enhanced delivery of thecorresponding prodrugs and for providing combination therapies includingthe prodrugs. The advantages for using prodrugs of the invention includethe facilitation of controlled formulation into an LNP of the disclosure(e.g., a liposome). This allows the prodrug to be maintained in aninactive form during systemic circulation, which allows the liposome torelease the active agent after engulfment by a cell, for example withina tumor.

In certain embodiments one or more TLR prodrugs (e.g., any one or moreof the TLR prodrugs inhibitors taught in Formula I or Formula II, and/orTR3, TR5, TR5(A), TR5(B), TR6, TR6(A)) (See, section entitled“Prodrugs”) are formulated a lipid moiety that forms a vesicle (e.g., aliposome) structure in aqueous solution or that can form a component ofa lipid bilayer comprising a liposome.

In certain embodiments one or more TLR Lipid Moieties (e.g., any one ormore of the TLR lipid moieties taught in Formula III or Formula IV,and/or TR8, TR11) (See, section entitled “Lipids”) are formulated and/orco-formulated within a vesicle (e.g., a liposome) structure in aqueoussolution or that can form a component of a lipid bilayer comprising aliposome.

The liposomes can be used directly or provided as components in acombined formulation (e.g., in combination with another drug moiety, orlipid moiety, or therapeutic modality as disclosed herein).

In certain embodiments, the liposome that is formulated with the TLRprodrug comprises a lipid, PHGP, vitamin E, cholesterol, and/or a fattyacid.

In certain embodiments, the liposome that is formulated comprises alipid moiety comprising TR8.

In certain embodiments, the liposome that is formulated comprises alipid moiety comprising TR11.

In certain embodiments, the liposome that is formulated comprises alipid moiety comprising Formula III.

In certain embodiments, the liposome that is formulated comprises alipid moiety comprising Formula IV.

In certain embodiments, the liposome that is formulated comprises alipid moiety comprising Alpha-galactosylceramide (α-GalCer).

In one embodiment, the liposome comprises cholesterol.

In one embodiment, the liposome comprises DPPG.

In one embodiment, the liposome comprises DMPG.

In one embodiment, the liposome Lyso PC.

In one embodiment, the liposome (Δ9-Cis) PG.

In one embodiment, the liposome comprises Soy Lyso PC.

In one embodiment, the liposome comprises PG.

In one embodiment, the liposome comprises PA-PEG3-mannose.

In one embodiment, the liposome comprises C16 PEG2000 Ceramide.

In one embodiment, the liposome comprises MPLA.

In one embodiment, the liposome comprises 3-Deacly MPLA.

In one embodiment, the liposome comprises CHEMS.

In one embodiment, the liposome comprises Stearic Acid.

In one embodiment, the liposome comprises a phospholipid set forth inTable III.

In one embodiment, the liposome comprises TR6 and further comprisesCHEMS and further comprises a LU wherein said LU is ahydromethylcarbamate linker.

In one embodiment, the liposome comprises TR6 and further comprisesStearic Acid and further comprises a LU wherein said LU is ahydromethylcarbamate linker.

In one embodiment, the liposome comprises TR6 and further comprisesCHEMS and further comprises a LU wherein said LU is ahydromethylcarbamate linker and further comprises a helper lipid setforth in Table II.

In one embodiment, the liposome comprises TR6 and further comprises aStearic Acid and further comprises a LU wherein said LU is ahydromethylcarbamate linker and further comprises a helper lipid setforth in Table II.

In one embodiment, the liposome comprises TR5.

In one embodiment, the liposome comprises TR5 and further comprisesCHEMS and further comprises a LU wherein said LU is ahydromethylcarbamate linker.

In one embodiment, the liposome comprises TR5 and further comprisesStearic Acid and further comprises a LU wherein said LU is ahydromethylcarbamate linker.

In one embodiment, the liposome comprises TR5 and further comprisesCHEMS and further comprises a LU wherein said LU is ahydromethylcarbamate linker and further comprises a helper lipid setforth in Table II.

In one embodiment, the liposome comprises TR5 and further comprises aStearic Acid and further comprises a LU wherein said LU is ahydromethylcarbamate linker and further comprises a helper lipid setforth in Table II.

In one embodiment, the liposome comprises TR5(B) and further comprisesCHEMS and further comprises a LU wherein said LU is ahydromethylcarbamate linker.

In one embodiment, the liposome comprises TR5(B) and further comprisesStearic Acid and further comprises a LU wherein said LU is ahydromethylcarbamate linker.

In one embodiment, the liposome comprises TR5(B) and further comprisesCHEMS and further comprises a LU wherein said LU is ahydromethylcarbamate linker and further comprises a helper lipid setforth in Table II.

In one embodiment, the liposome comprises TR5(B) and further comprises aStearic Acid and further comprises a LU wherein said LU is ahydromethylcarbamate linker and further comprises a helper lipid setforth in Table II.

In one embodiment, the liposome comprises TR3 and further comprisesCHEMS and further comprises a LU wherein said LU is ahydromethylcarbamate linker.

In one embodiment, the liposome comprises TR3 and further comprisesStearic Acid and further comprises a LU wherein said LU is ahydromethylcarbamate linker.

In one embodiment, the liposome comprises TR3 and further comprisesCHEMS and further comprises a LU wherein said LU is ahydromethylcarbamate linker and further comprises a helper lipid setforth in Table II.

In one embodiment, the liposome comprises TR3 and further comprises aStearic Acid and further comprises a LU wherein said LU is ahydromethylcarbamate linker and further comprises a helper lipid setforth in Table II.

In one embodiment, the liposome of the disclosure comprises a TLRprodrug co-formulated with one or more additional immune modulatingagents, whereby the immune modulating agents includes, but is notlimited to, immunogenic-cell death inducing chemotherapeutics, IDOantagonists, sting agonists, CTLA4 inhibitors, PD-1 inhibitors, and/orprodrugs thereof.

In one embodiment, the liposome of the disclosure comprises a TLRprodrug co-formulated with one or more additional immune modulatingagents, whereby the immune modulating agents includes, but is notlimited to, neurokinin 1 (NK1) antagonists, and/or prodrugs thereof.

In one embodiment, the liposome of the disclosure comprises a TLRprodrug co-formulated with one or more additional immune modulatingagents, whereby the immune modulating agents includes, but is notlimited to, A2aR antagonists, and/or prodrugs thereof.

In a preferred embodiment, the liposome comprises a TLR prodrugco-formulated with an ICD-inducing Chemotherapeutic.

In a preferred embodiment, the liposome comprises a TLR prodrugco-formulated with an ICD-inducing Chemotherapeutic selected from thelist: doxorubicin (DOX), mitoxantrone (MTO), Oxaliplatin (OXA),Cyclophosphamide (CP), Bortezomib, Carfilzimib, or Paclitaxel.

In a preferred embodiment, the liposome comprises a TLR prodrugco-formulated with a Toll Receptor TLR agonist/Prodrug.

In a preferred embodiment, the liposome comprises a TLR prodrugco-formulated with Toll Receptor (TLR) agonist/Prodrug selected from thelist: Resiquimod (R848), Gardiquimod, 852A, DSR 6434, Telratolimod,CU-T12-9, monophosphoryl Lipid A (MPLA), 3D(6-acyl)-PHAD®, SMU127,Pam3CSK4, or 3D-PHAD®.

In a preferred embodiment, the liposome comprises a TLR prodrugco-formulated with an PD-1 inhibitor/Prodrug.

In a preferred embodiment, the liposome comprises a TLR prodrugco-formulated with an PD-1 inhibitor/Prodrug, selected from the list:AUNP12, CA-170, or BMS-986189 or prodrugs thereof.

In a preferred embodiment, the liposome comprises a TLR prodrugco-formulated with doxorubicin (DOX).

In a preferred embodiment, the liposome comprises a TLR prodrugco-formulated with mitoxantrone (MTO).

In a preferred embodiment, the liposome comprises a TLR prodrugco-formulated with doxorubicin (DOX) and an PD-1 prodrug.

In a preferred embodiment, the liposome comprises a TLR prodrugco-formulated with mitoxantrone (MTO) and a PD-1 prodrug.

In a preferred embodiment, the liposome comprises a TLR prodrugco-formulated with doxorubicin (DOX) and an IDO antagonist/prodrug.

In a preferred embodiment, the liposome comprises a TLR prodrugco-formulated with mitoxantrone (MTO) and an IDO antagonist/prodrug.

In a preferred embodiment, the liposome comprises a TLR prodrugco-formulated with doxorubicin (DOX) and a PD-1 prodrug and an IDOantagonist/prodrug.

In a preferred embodiment, the liposome comprises a TLR prodrugco-formulated with mitoxantrone (MTO) and a PD-1 prodrug and an IDOantagonist/prodrug.

In a preferred embodiment, the liposome comprises a TLR prodrugco-formulated with an IDO antagonist/prodrug.

In a preferred embodiment, the liposome comprises a TLR prodrugco-formulated with an IDO antagonist/prodrug.

In a preferred embodiment, the liposome comprises a TLR prodrugco-formulated with an IDO antagonist/prodrug and a PD-1 prodrug.

In a preferred embodiment, the liposome comprises a TLR prodrugco-formulated with an IDO antagonist/prodrug and a PD-1 prodrug.

In a preferred embodiment, the liposome comprises TR6 co-formulated withdoxorubicin (DOX).

In a preferred embodiment, the liposome comprises TR6 co-formulated withmitoxantrone (MTO).

In a preferred embodiment, the liposome comprises TR6 co-formulated withdoxorubicin (DOX) and/or an IDO prodrug and/or an IDOantagonist/prodrug.

In a preferred embodiment, the liposome comprises TR6 co-formulated withmitoxantrone (MTO) and/or an IDO prodrug and/or an IDOantagonist/prodrug.

In a preferred embodiment, the liposome comprises TR6 co-formulated withNK1.

In a preferred embodiment, the liposome comprises TR6 co-formulated withMTO and TR8.

In a preferred embodiment, the liposome comprises TR6 co-formulated withDOX and a A2aR prodrug.

In a preferred embodiment, the liposome comprises TR5 co-formulated withDOX.

In a preferred embodiment, the liposome comprises TR5 co-formulated witha A2aR prodrug.

In a preferred embodiment, the liposome comprises TR5 co-formulated withDOX and a A2aR prodrug.

In a preferred embodiment, the liposome comprises TR5 co-formulated withan A2aR prodrug and an anti-PD1 antibody.

In a preferred embodiment, the liposome comprises TR5 co-formulated withan A2aR prodrug and an IDO prodrug.

In a preferred embodiment, the liposome comprises TR5(B) co-formulatedwith doxorubicin (DOX).

In a preferred embodiment, the liposome comprises TR5(B) co-formulatedwith mitoxantrone (MTO).

In a preferred embodiment, the liposome comprises TR5(B) co-formulatedwith doxorubicin (DOX) and/or an IDO prodrug and/or an IDOantagonist/prodrug.

In a preferred embodiment, the liposome comprises TR5(B) co-formulatedwith mitoxantrone (MTO) and/or an IDO prodrug and/or an IDOantagonist/prodrug.

In a preferred embodiment, the liposome comprises TR8 co-formulated withdoxorubicin (DOX).

In a preferred embodiment, the liposome comprises TR8 co-formulated withmitoxantrone (MTO).

In a preferred embodiment, the liposome comprises TR8 co-formulated withdoxorubicin (DOX) and/or a TLR prodrug and/or an IDO antagonist/prodrug.

In a preferred embodiment, the liposome comprises TR8 co-formulated withmitoxantrone (MTO) and/or a TLR prodrug and/or an IDOantagonist/prodrug.

In a preferred embodiment, the liposome comprises TR11 co-formulatedwith doxorubicin (DOX).

In a preferred embodiment, the liposome comprises TR11 co-formulatedwith mitoxantrone (MTO).

In a preferred embodiment, the liposome comprises TR11 co-formulatedwith doxorubicin (DOX) and/or a TLR prodrug and/or an IDOantagonist/prodrug.

In a preferred embodiment, the liposome comprises TR11 co-formulatedwith mitoxantrone (MTO) and/or a TLR prodrug and/or an IDOantagonist/prodrug.

In another preferred embodiment, the liposome comprises a solid-lipidnanoparticle (SLNP) comprising a liposome which comprises a TLR prodrug.

One of skill in the art will appreciate and understand that solubilityis one of most common problems faced by the artisan in the drugdevelopment process. Chemical conjugation of a drug/anti-cancer agentsvia lipid molecules (i.e., lipid-based prodrugs) provides a platform tosolve the problem of formulating the drugs in an aqueous suspension. Themajor advantages of delivering drug(s) with lipid conjugation(lipid-based prodrugs) lies on its ability to improvepharmacokinetics/half-life and targeted delivery.

With suitable selection of lipid molecules, lipid-based prodrug(s) canbe integrated/formulated in a liposomal formulation using techniquesknown in the art, which has many more advantages over conventional drugdelivery system. (KOHLI, et. al., J. Control Release, 0: pp 274-287(Sept. 28, 2014); and GARCIA-PINEL, et. al., Nanomaterials 9:638 (2019).The advantage of combining lipid-prodrug with liposomes is twofold: (i)liposomes containing lipid-prodrug not only increase the solubility ofthe drug/prodrug itself, but (ii) also have the ability to encapsulatemultiple drugs (both hydrophilic and lipophilic) (see, section entitlednanocarriers).

For the purposes of this disclosure, the major advantage of liposomeformulations are as follows:

i) biocompatibility/biodegradability and no general toxicity of theliposome's formulations;

ii) flexibility and manipulation of size and surface charge depending onthe required purpose. Liposome formulation(s), for the purposes of thisdisclosure, can have a size range of 40-150 nm in diameter and a surfacecharge in the range of −40 to +40 mV; and

iii) Liposomes of the invention have either a single or multiplelipid-prodrugs as the constituent lipid portion of the liposome(s).Additionally, multiple drugs (e.g., that work in different mechanism ofaction) and with different solubility profile (hydrophilic orlipophilic) can be formulated (either in the lipid bilayers or in thehydrophilic core) in these liposomes.

As one of ordinary skill in the art will appreciate, all methods ofmaking liposomes involve four (4) basic stages:

(i) Drying down lipids from organic solvent;

(ii) Dispersing the lipid in aqueous solution;

(iii) Purifying the resultant liposome; and

(iv) Analyzing the final product.

See, AKBARZADEH, et. al., Nanoscale Research Letters, 8:102 (2013).

Another aspect of the invention discloses liposomal encapsulationtechnology (LET) which is a delivery technique used to transmit drugs.LET is a method of generating sub-microscopic foams called liposomes,which encapsulate numerous materials. These ‘liposomes’ form a barrieraround their contents, which is resistant to enzymes in the mouth andstomach, alkaline solutions, digestive juices, bile salts, andintestinal flora that are generated in the human body, as well as freeradicals. The contents of the liposomes are, therefore, protected fromoxidation and degradation. This protective phospholipid shield orbarrier remains undamaged until the contents of the liposome aredelivered to the exact target gland, organ, or system where the contentswill be utilized (See, section entitled nanocarriers).

In one embodiment, liposome(s) of the disclosure are synthesized using aplurality of different ratios of TLR prodrugs, TLR lipid moieties,lipids, and/or lipid-prodrugs. As disclosed herein, the TLR prodrugs maycomprise helper lipids as disclosed herein (See, for example Table II).

In one embodiment, liposome(s) of the disclosure are synthesized using aplurality of different ratios of TLR prodrugs, TLR lipid moieties,lipids, and/or lipid-prodrugs. As disclosed herein, the TLR prodrugs mayfurther comprise DSPE-PEGs.

In a preferred embodiment, the liposomes of the invention comprise acomposition having the following ratio(s):

Constituent of the Liposome Amount (% w/w) Lipid 1 (lipid-prodrug) 5-60Lipid 2 (lipid-prodrug) 0-60 Helper lipids 0-50 DSPEG-PEG 2000 2-5

In a further preferred embodiment, the liposomes of the inventioncomprise a composition having the following ratio(s):

Constituent of the Liposome Amount (% w/w) Lipid 1 (lipid-prodrug) 5-60Helper lipids 0-50 DSPEG-PEG 2000 2-5

In a further preferred embodiment, the liposomes of the inventioncomprise a composition having the following ratio(s):

Constituent of the Liposome Amount (% w/w) Lipid 1 (lipid-prodrug) 5-60Helper lipids 0-50 DSPEG-PEG 2000 2-5Whereby Lipid 1 comprises TR5 and CHEMS.

In a further preferred embodiment, the liposomes of the inventioncomprise a composition having the following ratio(s):

Constituent of the Liposome Amount (% w/w) Lipid 1 (lipid-prodrug) 5-60Helper lipids 0-50 DSPEG-PEG 2000 2-5Whereby Lipid 1 comprises TR5 and Stearic Acid.

In a further preferred embodiment, the liposomes of the inventioncomprise a composition having the following ratio(s):

Constituent of the Liposome Amount (% w/w) Lipid 1 (lipid-prodrug) 5-60Helper lipids 0-50 DSPEG-PEG 2000 2-5Whereby Lipid 1 comprises TR6 and CHEMS.

In a further preferred embodiment, the liposomes of the inventioncomprise a composition having the following ratio(s):

Constituent of the Liposome Amount (% w/w) Lipid 1 (lipid-prodrug) 5-60Helper lipids 0-50 DSPEG-PEG 2000 2-5Whereby Lipid 1 comprises TR6 and Stearic Acid.

In a further preferred embodiment, the liposomes of the inventioncomprise a composition having the following ratio(s):

Constituent of the Liposome Amount (% w/w) Lipid 1 (lipid-prodrug) 5-60Helper lipids 0-50 DSPEG-PEG 2000 2-5Whereby Lipid 1 comprises TR3 and Stearic Acid.

One of ordinary skill in the art will appreciate and be enabled to makevariations and modifications to the disclosed embodiment withoutaltering the function and purpose of the invention disclosed herein.Such variations and modifications are intended within the scope of thepresent disclosure.

VIII.) Pharmaceutical Formulation

As used herein, the term “drug” is synonymous with “pharmaceutical”. Incertain embodiments, the liposome of the disclosure is fabricated to anencapsulated dosage form to and given to a patient for the treatment ofdisease.

Generally speaking, pharmaceutical formulation is the process in whichdifferent chemical substances are combined to a pure drug substance toproduce a final drug product. Formulation studies involve developing apreparation of the drug which is both stable and acceptable to thepatient. For orally taken drugs, this usually involves incorporating thedrug into a tablet or a capsule. It is important to appreciate that adosage form contains a variety of other substances apart from the drugitself, and studies have to be carried out to ensure that the drug iscompatible with these other substances.

An excipient is an inactive substance used as a carrier for the activeingredients of a drug product, in this case a liposome comprising a TLRprodrug. In addition, excipients can be used to aid the process by whicha drug product is manufactured. The active substance is then dissolvedor mixed with an excipient. Excipients are also sometimes used to bulkup formulations with very potent active ingredients, to allow forconvenient and accurate dosage. Once the active ingredient has beenpurified, it cannot stay in purified form for an extended amount oftime. In many cases it will denature, fall out of solution, or stick tothe sides of the container.

To stabilize the active ingredient, excipients are added to ensure thatthe active ingredient stays active and is stable for a long enoughperiod of time that the shelf-life of the product makes it competitivewith other products and safe for the end-user. Examples of excipientsinclude but are not limited to, anti-adherents, binders, coatings,disintegrants, fillers, diluents, flavors, colors, lubricants, andpreservatives. The final formulation comprises and active ingredient andexcipients which are then enclosed in the pharmaceutical dosage form.

Pre-formulation involves the characterization of a drug's physical,chemical, and mechanical properties in order to choose what otheringredients should be used in the preparation. Formulation studies thenconsider such factors as stability, particle size, polymorphism, pH, andsolubility, as all of these can influence bioavailability and hence theactivity of a drug. The drug must be combined with inactive additives bya method which ensures that the quantity of drug present is consistentin each dosage unit (e.g., each vial). The dosage should have a uniformappearance.

It is unlikely that these studies will be complete by the time clinicaltrials commence. This means that simple preparations are developedinitially for use in phase I clinical trials. These typically consist ofvials, hand-filled capsules containing a small amount of the drug and adiluent. Proof of the long-term stability of these formulations is notrequired, as they will be used (tested) in a matter of days. However,long-term stability is critical in supply chain management since thetime the final formulation is packaged until it reaches the patient canbe several months or years. Consideration has to be given to what iscalled the drug load (i.e., the ratio of the active drug to the totalcontents of the dose). A low drug load may cause homogeneity problems. Ahigh drug load may pose flow problems or require large capsules if thecompound has a low bulk density. By the time phase III clinical trialsare reached, the formulation of the drug should have been developed tobe close to the preparation that will ultimately be used in the market.

A knowledge of stability is essential by this stage, and conditions musthave been developed to ensure that the drug is stable in thepreparation. If the drug proves unstable, it will invalidate the resultsfrom clinical trials since it would be impossible to know what theadministered dose actually was. Stability studies are carried out totest whether temperature, humidity, oxidation, or photolysis(ultraviolet light or visible light) have any effect, and thepreparation is analyzed to see if any degradation products have beenformed. It is also important to check whether there are any unwantedinteractions between the preparation and the container. If a plasticcontainer is used, tests are carried out to see whether any of theingredients become adsorbed on to the plastic, and whether anyplasticizers, lubricants, pigments, or stabilizers leach out of theplastic into the preparation. Even the adhesives for the container labelneed to be tested, to ensure they do not leach through the plasticcontainer into the preparation. The way a drug is formulated can avoidsome of the problems associated with oral administration. Drugs arenormally taken orally as tablets or capsules. The drug (activesubstance) itself needs to be soluble in aqueous solution at acontrolled rate. Such factors as particle size and crystal form cansignificantly affect dissolution. Fast dissolution is not always ideal.For example, slow dissolution rates can prolong the duration of actionor avoid initial high plasma levels.

In some embodiments, the nanocarrier (e.g., a liposome comprising a TLRprodrug) and/or the liposome comprising a TLR prodrug and co-formulatedwith an immune modulating agent are administered alone or in a mixturewith a physiologically-acceptable carrier (such as physiological salineor phosphate buffer) selected in accordance with the route ofadministration and standard pharmaceutical practice. For example, whenused as an injectable, the nanocarriers can be formulated as a sterilesuspension, dispersion, or emulsion with a pharmaceutically acceptablecarrier. In certain embodiments normal saline can be employed as thepharmaceutically acceptable carrier. Other suitable carriers include,e.g., water, buffered water, 0.4% saline, 0.3% glycine, 5% glucose andthe like, including glycoproteins for enhanced stability, such asalbumin, lipoprotein, globulin, etc. In compositions comprising salineor other salt-containing carriers, the carrier is preferably addedfollowing nanocarrier formation. Thus, after the nanocarrier is formedand loaded with suitable drug(s), the nanocarrier can be diluted intopharmaceutically acceptable carriers such as normal saline. Similarly,the TLR prodrug liposomes can be introduced into carriers thatfacilitate suspension of the nanomaterials (e.g., emulsions, dilutions,etc.).

The pharmaceutical compositions may be sterilized by conventional,well-known sterilization techniques. The resulting aqueous solutions,suspensions, dispersions, emulsions, etc., may be packaged for use orfiltered under aseptic conditions. In certain embodiments the drugdelivery nanocarriers (e.g., LB-coated nanoparticles) are lyophilized,the lyophilized preparation being combined with a sterile aqueoussolution prior to administration. The compositions may also containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH-adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, etc.

Additionally, in certain embodiments, the pharmaceutical formulation mayinclude lipid-protective agents that protect lipids against free-radicaland lipid-peroxidative damage on storage. Lipophilic free-radicalquenchers, such as alpha-tocopherol and water-soluble iron-specificchelators, such as ferrioxamine, are suitable and contemplated herein.The concentration of nanocarrier (e.g., liposome comprising TLRprodrugs) in the pharmaceutical formulations can vary widely, e.g., fromless than approximately 0.05%, usually at least approximately 2 to 5% toas much as 10 to 50%, or to 40%, or to 30% by weight and are selectedprimarily by fluid volumes, viscosities, etc., in accordance with theparticular mode of administration selected. For example, theconcentration may be increased to lower the fluid load associated withtreatment. This may be particularly desirable in patients havingatherosclerosis-associated congestive heart failure or severehypertension. Alternatively, nanocarriers composed of irritating lipidsmay be diluted to low concentrations to lessen inflammation at the siteof administration. The amount of nanocarriers administered will dependupon the particular drug used, the disease state being treated and thejudgment of the clinician but will generally be between approximately0.01 and approximately 50 mg per kilogram of body weight, preferablybetween approximately 0.1 and approximately 5 mg per kg of body weight.

One of skill in the art will appreciate that exact dosages will varydepending upon such factors as the particular TLR prodrugs and anyco-formulated immune modulating agents and the desirable medical effect,as well as patient factors such as age, sex, general condition, and thelike. Those of skill in the art can readily take these factors intoaccount and use them to establish effective therapeutic concentrationswithout resort to undue experimentation.

For administration to humans (or to non-human mammals) in the curative,remissive, retardive, or prophylactic treatment of diseases describedherein the prescribing physician will ultimately determine theappropriate dosage of the drug for a given human (or non-human) subject,and this can be expected to vary according to the age, weight, andresponse of the individual as well as the nature and severity of thepatient's disease. In certain embodiments the dosage of the drugprovided by the nanocarrier(s) can be approximately equal to thatemployed for the free drug. However as noted above, the nanocarriersdescribed herein can significantly reduce the toxicity of the drug(s)administered thereby and significantly increase a therapeutic window.Accordingly, in some cases dosages in excess of those prescribed for thefree drug(s) will be utilized.

One of ordinary skill in the art will appreciate and be enabled to makevariations and modifications to the disclosed embodiment withoutaltering the function and purpose of the invention disclosed herein.Such variations and modifications are intended within the scope of thepresent disclosure.

IX.) Combination Therapy

As the skilled artisan will appreciate and understand, cancer cellgrowth and survival can be impacted by multiple signaling pathways.Thus, it is useful to combine different enzyme/protein/receptorinhibitors, exhibiting different preferences in the targets which theymodulate the activities of, to treat such conditions. Targeting morethan one signaling pathway (or more than one biological moleculeinvolved in a given signaling pathway) may reduce the likelihood ofdrug-resistance arising in a cell population, and/or reduce the toxicityof treatment.

Thus, the liposomes comprising TLR prodrugs of the present disclosurecan be used in combination with one or more otherenzyme/protein/receptor inhibitors or one or more therapies for thetreatment of diseases, such as cancer or infections. Examples ofdiseases and indications treatable with combination therapies includethose set forth in the present disclosure. Examples of cancers include,but are not limited to, solid tumors and liquid tumors, such as bloodcancers. Examples of infections include viral infections, bacterialinfections, fungus infections or parasite infections.

For example, the liposomes comprising TLR prodrugs of the presentdisclosure can be combined with one or more inhibitors of the followingkinases for the treatment of cancer: Akt1, Akt2, Akt3, TGF-βR, PKA, PKG,PKC, CaM-kinase, phosphorylase kinase, MEKK, ERK, MAPK, mTOR, EGFR,HER2, HER3, HER4, INS-R, IGF-1R, IR-R, PDGFαR, PDGFβR, PI3K (alpha,beta, gamma, delta), CSFIR, KIT, FLK-II, KDR/FLK-1, FLK-4, flt-1, FGFR1,FGFR2, FGFR3, FGFR4, c-Met, Ron, Sea, TRKA, TRKB, TRKC, TAM kinases(Axl, Mer, Tyro3), FLT3, VEGFR/Flt2, Flt4, EphA1, EphA2, EphA3, EphB2,EphB4, Tie2, Src, Fyn, Lck, Fgr, Btk, Fak, SYK, FRK, JAK, ABL, ALK andB-Raf.

In further embodiments, the liposomes comprising TLR prodrugs of thepresent disclosure can be combined with one or more of the followinginhibitors for the treatment of cancer or infections. Non-limitingexamples of inhibitors that can be combined with the compounds of thepresent disclosure for treatment of cancer and infections include anFGFR inhibitor (FGFR1, FGFR2, FGFR3 or FGFR4, e.g., INCB54828, INCB62079and INCB63904), a JAK inhibitor (JAK1 and/or JAK2, e.g., ruxolitinib,baricitinib or INCB39110), a TLR inhibitor (e.g., epacadostat, NLG919,or BMS-986205), an LSD1 inhibitor (e.g., INCB59872 and INCB60003), a TDOinhibitor, a PI3K-delta inhibitor (e.g., INCB50797 and INCB50465), aPI3K-gamma inhibitor such as PI3K-gamma selective inhibitor, a Piminhibitor (e.g., INCB53914), a CSF1R inhibitor, a TAM receptor tyrosinekinases (Tyro-3, Axl, and Mer), an adenosine receptor antagonist (e.g.,A2a/A2b receptor antagonist), an HPK1 inhibitor, a histone deacetylaseinhibitor (HDAC) such as an HDAC8 inhibitor, an angiogenesis inhibitor,an interleukin receptor inhibitor, bromo and extra terminal familymembers inhibitors (for example, bromodomain inhibitors or BETinhibitors such as INCB54329 and INCB57643), a poly ADP ribosepolymerase (PARP) inhibitor such as rucaparib, olaparib, niraparib,veliparib, or talazoparib, an arginase inhibitor (INCB01158), a PD-1inhibitor, a PD-1/L-1 inhibitor, a PD-1/L-2 inhibitor, and an adenosinereceptor antagonist or combinations thereof.

In further embodiments, the liposomes comprising TLR prodrugs of thepresent disclosure can be combined with one or more activator ofinvariant natural killer T (iNKT) cells including but not limited to,α-galactosylceramida (α-GalCer) and analogs thereof including, C8Galactosyl(α) Ceramide, C16 Galactosyl(α) Ceramide, and C24:1Galactosyl(α) Ceramide (Avanti Polar Lipids, Alabaster, Alabama).

Additionally, the liposomes comprising TLR prodrugs of the presentdisclosure can further be used in combination with other methods oftreating cancers, for example by chemotherapy, irradiation therapy,tumor-targeted therapy, adjuvant therapy, immunotherapy or surgery.

Examples of immunotherapy include cytokine treatment (e.g., interferons,GM-CSF, G-CSF, IL-2), CRS-207 immunotherapy, cancer vaccine, monoclonalantibody, adoptive T cell transfer, Toll receptor agonists, STINGagonists, oncolytic virotherapy and immunomodulating small molecules,including thalidomide or JAK1/2 inhibitor and the like.

The liposomes comprising TLR prodrugs can be administered in combinationwith one or more anti-cancer drugs, such as a chemotherapeutics. Examplechemotherapeutics include any of: abarelix, aldesleukin, alemtuzumab,alitretinoin, allopurinol, altretamine, anastrozole, arsenic trioxide,asparaginase, azacitidine, bevacizumab, bexarotene, baricitinib,bleomycin, bortezombi, bortezomib, busulfan intravenous, busulfan oral,calusterone, capecitabine, carboplatin, carmustine, cetuximab,chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide,cytarabine, dacarbazine, dactinomycin, dalteparin sodium, dasatinib,daunorubicin, decitabine, denileukin, denileukin diftitox, dexrazoxane,docetaxel, doxorubicin, dromostanolone propionate, eculizumab,epirubicin, erlotinib, estramustine, etoposide phosphate, etoposide,exemestane, fentanyl citrate, filgrastim, floxuridine, fludarabine,fluorouracil, fulvestrant, gefitinib, gemcitabine, gemtuzumabozogamicin, goserelin acetate, histrelin acetate, ibritumomab tiuxetan,idarubicin, ifosfamide, imatinib mesylate, interferon alfa 2a,irinotecan, lapatinib ditosylate, lenalTLRmide, letrozole, leucovorin,leuprolide acetate, levamisole, lomustine, meclorethamine, megestrolacetate, melphalan, mercaptopurine, methotrexate, methoxsalen, mitomycinC, mitotane, mitoxantrone, nandrolone phenpropionate, nelarabine,nofetumomab, olaparib, oxaliplatin, paclitaxel, pamidronate,panitumumab, pegaspargase, pegfilgrastim, pemetrexed disodium,pentostatin, pipobroman, plicamycin, procarbazine, quinacrine,rasburicase, rituximab, ruxolitinib, rucaparib, sorafenib, streptozocin,sunitinib, sunitinib maleate, tamoxifen, temozolomide, teniposide,testolactone, thalTLRmide, thioguanine, thiotepa, topotecan, toremifene,tositumomab, trastuzumab, tretinoin, uracil mustard, valrubicin,vinblastine, vincristine, vinorelbine, vorinostat, niraparib, veliparib,talazoparib and zoledronate.

Other anti-cancer agent(s) include antibody therapeutics such astrastuzumab (Herceptin), antibodies to costimulatory molecules such asCTLA-4 (e.g., ipilimumab), 4-1BB (e.g., urelumab, utomilumab),antibodies to PD-1 and PD-L1/L2, or antibodies to cytokines (IL-10,TGF-.beta., etc.).

Examples of antibodies to PD-1 and/or PD-L1/L2 that can be combined withcompounds of the present disclosure for the treatment of cancer orinfections such as viral, bacteria, fungus and parasite infectionsinclude, but are not limited to, nivolumab, pembrolizumab, MPDL3280A,MEDI-4736 and SHR-1210.

In addition, liposomes comprising TLR prodrugs of the present disclosurecan be used in combination with one or more immune checkpoint inhibitorsfor the treatment of diseases, such as cancer or infections. Exemplaryimmune checkpoint inhibitors include inhibitors against immunecheckpoint molecules such as CD27, CD28, CD40, CD122, CD96, CD73, CD47,OX40, GITR, CSF1R, JAK, P13K delta, P13K gamma, TAM, arginase, CD137(also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3,TIM3, VISTA, PD-1, PD-L1 and PD-L2

In some embodiments, the immune checkpoint molecule is a stimulatorycheckpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR andCD137. In further embodiments, the immune checkpoint molecule is aninhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA,CTLA-4, TLR, KIR, LAG3, PD-1, TIM3, and VISTA. In further embodiments,the liposomes comprising TLR prodrugs provided herein can be used incombination with one or more agents selected from KIR inhibitors, TIGITinhibitors, LAIR1 inhibitors, CD160 inhibitors, 2B4 inhibitors and TGFRbeta inhibitors.

X.) Methods of Delivering Liposomes Comprising TLR Prodrugs to a CellExpressing Toll-Like Receptor (“TLR”)

As it is known in the art, a wide variety of compositions and methodsfor using prodrugs and/or liposomes to kill tumor cells are known in theart. In the context of cancers, typical methods entail administering toa mammal having a tumor, a biologically effective amount of a TLRprodrug of the disclosure, and/or a liposome of the disclosurecomprising a TLR prodrug.

A typical embodiment is a method of delivering a therapeutic agent to acell expressing TLR1/2, TLR4, TLR7, TLR8, and/or TLR7/8, comprisingforming a TLR prodrug by conjugating a drug moiety of the disclosurewith a lipid of the disclosure via a Linkage Unit, and exposing the cellto the TLR prodrug.

In one embodiment, the TLR prodrug comprises a drug moiety of Formula Iand CHEMS conjugated via a LU comprising a hydromethylcarbamate linker.

In one embodiment, the TLR prodrug comprises a drug moiety of Formula Iand Stearic Acid conjugated via a LU comprising a hydromethylcarbamatelinker.

In one embodiment, the TLR prodrug comprises a drug moiety of Formula IIand CHEMS conjugated via a LU comprising a hydromethylcarbamate linker.

In one embodiment, the TLR prodrug comprises a drug moiety of Formula IIand Stearic Acid conjugated via a LU comprising a hydromethylcarbamatelinker.

In one embodiment, the TLR prodrug comprises TR3 and CHEMS conjugatedvia a LU comprising a hydromethylcarbamate linker.

In one embodiment, the TLR prodrug comprises TR3 and Stearic Acidconjugated via a LU comprising a hydromethylcarbamate linker.

In one embodiment, the TLR prodrug comprises TR6 and CHEMS conjugatedvia a LU comprising a hydromethylcarbamate linker.

In one embodiment, the TLR prodrug comprises TR6 and Stearic Acidconjugated via a LU comprising a hydromethylcarbamate linker.

In one embodiment, the TLR prodrug comprises TR5(B) and CHEMS conjugatedvia a LU comprising a hydromethylcarbamate linker.

In one embodiment, the TLR prodrug comprises TR5(B) and Stearic Acidconjugated via a LU comprising a hydromethylcarbamate linker.

Another illustrative embodiment is a method of treating an individualsuspected of suffering from metastasized cancer, comprising a step ofadministering parenterally to said individual a pharmaceuticalcomposition comprising a therapeutically effective amount of a TLRprodrug produced by conjugating a drug moiety with a lipid of thedisclosure via a Linkage Unit, and exposing the cell to the TLR prodrug.

In one embodiment, the TLR prodrug comprises a drug moiety of Formula Iand CHEMS conjugated via a LU comprising a hydromethylcarbamate linker.

In one embodiment, the TLR prodrug comprises a drug moiety of Formula Iand Stearic Acid conjugated via a LU comprising a hydromethylcarbamatelinker.

In one embodiment, the TLR prodrug comprises a drug moiety of Formula IIand CHEMS conjugated via a LU comprising a hydromethylcarbamate linker.

In one embodiment, the TLR prodrug comprises a drug moiety of Formula IIand Stearic Acid conjugated via a LU comprising a hydromethylcarbamatelinker.

In one embodiment, the TLR prodrug comprises TR3 and CHEMS conjugatedvia a LU comprising a hydromethylcarbamate linker.

In one embodiment, the TLR prodrug comprises TR3 and Stearic Acidconjugated via a LU comprising a hydromethylcarbamate linker.

In one embodiment, the TLR prodrug comprises TR6 and CHEMS conjugatedvia a LU comprising a hydromethylcarbamate linker.

In one embodiment, the TLR prodrug comprises TR6 and Stearic Acidconjugated via a LU comprising a hydromethylcarbamate linker.

In one embodiment, the TLR prodrug comprises TR5(B) and CHEMS conjugatedvia a LU comprising a hydromethylcarbamate linker.

In one embodiment, the TLR prodrug comprises TR5(B) and Stearic Acidconjugated via a LU comprising a hydromethylcarbamate linker.

TLR prodrugs, liposomes, and co-formulated liposomes of the presentdisclosure inhibit the activity of TLR protein/protein interaction and,thus, are useful in treating diseases and disorders associated withactivity of TLR and the diseases and disorders.

In further embodiments of the disclosure, the TLR prodrugs, liposomes,or pharmaceutically acceptable salts or stereoisomers thereof, areuseful for therapeutic administration to enhance, stimulate and/orincrease immunity in cancer, chronic infection, or sepsis, includingenhancement of response to vaccination.

In further embodiments, the present disclosure provides a method forinhibiting the TLR (e.g., TLR1/2, TLR4, TLR7, TLR8, and/or TLR7/8)T-cell function. The method includes administering to an individual or apatient a TLR prodrug, liposomes, and/or of any of the formulas asdescribed herein (e.g., TR3, TR6, TR6(A), TR5, TR5(A) and/or TR5(B)), orof a TLR prodrug, liposomes, and nano-encapsulated TLR inhibitorprodrugs as recited in any of the claims and described herein, or apharmaceutically acceptable salt or a stereoisomer thereof. The TLRprodrug, liposomes, and nano-encapsulated TLR inhibitor prodrugs of thepresent disclosure can be used alone, in combination with other agentsor therapies or as an adjuvant or neoadjuvant for the treatment ofdiseases or disorders, including cancer and other diseases. For the usesand methods described herein, any of the TLR prodrugs, liposomes, andnano-encapsulated TLR prodrugs of the disclosure, including any of theembodiments thereof, may be used.

In addition, The TLR prodrugs, liposomes, and nano-encapsulated TLRinhibitor prodrugs of the present disclosure inhibit the TLR function,resulting in a TLR pathway blockade.

In further embodiments, the present disclosure provides treatment of anindividual or a patient in vivo using TLR prodrugs, liposomes, andnano-encapsulated TLR inhibitor prodrug or a salt or stereoisomerthereof such that growth of cancerous tumors is inhibited.

TLR prodrugs, liposomes, and nano-encapsulated TLR inhibitor prodrugs,or of any of the formulas as described herein (e.g., TR3, TR6, TR6(A),TR5, TR5(A) and/or TR5(B)), or TLR prodrugs, liposomes, andnano-encapsulated TLR inhibitor prodrugs as recited in any of the claimsand described herein, or a salt or stereoisomer thereof, can be used toinhibit the growth of cancerous tumors.

In the alternative, TLR prodrugs, liposomes, and nano-encapsulated TLRprodrugs of the disclosure, or of any of the formulas as describedherein, or a compound as recited in any of the claims and describedherein (e.g., TR3, TR6, TR6(A), TR5, TR5(A) and/or TR5(B)), or a salt orstereoisomer thereof, can be used in conjunction with other agents orstandard cancer treatments, as described in this disclosure.

In a further embodiment, the present disclosure provides a method forinhibiting growth of tumor cells in vitro. The method includescontacting the tumor cells in vitro with TLR prodrugs, liposomes, andnano-encapsulated TLR inhibitor prodrugs of the disclosure, or of any ofthe formulas as described herein (e.g., TR3, TR6, TR6(A), TR5, TR5(A)and/or TR5(B)), or of a TLR prodrug, liposomes, and nano-encapsulatedTLR inhibitor prodrugs as recited in any of the claims and describedherein, or of a salt or stereoisomer thereof.

In a further embodiment, the present disclosure provides a method forinhibiting growth of tumor cells in a patient. The method includescontacting the tumor cells with TLR prodrugs, liposomes, andnano-encapsulated TLR inhibitor prodrugs of the disclosure, or of any ofthe formulas as described herein (e.g., TR3, TR6, TR6(A), TR5, TR5(A)and/or TR5(B)), or of a TLR prodrug, liposomes, and nano-encapsulatedTLR inhibitor prodrugs as recited in any of the claims and describedherein, or of a salt or stereoisomer thereof.

XI.) Methods of Treating Cancer(s) and Other Immunological Disorder(s)

Another embodiment of the present disclosure is a method for treatingcancer. The method comprises administering to a patient, atherapeutically effective amount of a liposome comprising a TLR prodrug(i.e., TR3, TR6, TR6(A), TR5, TR5(A) and/or TR5(B)) herein, a compoundas recited in any of the claims and described herein, or a salt thereof.Examples of cancers include those whose growth may be inhibited usingTLR inhibitors of the disclosure and TLR prodrugs of the disclosure andcancers typically responsive to immunotherapy.

In some embodiments, the present disclosure provides a method ofenhancing, stimulating and/or increasing the immune response in apatient. The method includes administering to the patient atherapeutically effective amount of a TLR prodrug and/or a liposomecomprising the same (i.e., TR3, TR6, TR6(A), TR5, TR5(A) and/or TR5(B)),a compound or composition as recited in any of the claims and describedherein, or a salt thereof.

Non-limiting examples of cancers that are treatable using the liposomescomprising TLR prodrugs, TLR prodrugs and co-formulated liposomes of thepresent disclosure include, but are not limited to, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular malignant melanoma, uterine cancer, ovarian cancer, rectalcancer, cancer of the anal region, stomach cancer, testicular cancer,uterine cancer, carcinoma of the fallopian tubes, carcinoma of theendometrium, endometrial cancer, carcinoma of the cervix, carcinoma ofthe vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin'slymphoma, cancer of the esophagus, cancer of the small intestine, cancerof the endocrine system, cancer of the thyroid gland, cancer of theparathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue,cancer of the urethra, cancer of the penis, chronic or acute leukemiasincluding acute myeloid leukemia, chronic myeloid leukemia, acutelymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors ofchildhood, lymphocytic lymphoma, cancer of the bladder, cancer of thekidney or urethra, carcinoma of the renal pelvis, neoplasm of thecentral nervous system (CNS), primary CNS lymphoma, tumor angiogenesis,spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi'ssarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally induced cancers including those induced by asbestos, andcombinations of said cancers. The compounds of the present disclosureare also useful for the treatment of metastatic cancers, especiallymetastatic cancers that express TLR.

In some embodiments, cancers treatable with liposomes, or TLR prodrugsof the present disclosure include melanoma (e.g., metastatic malignantmelanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer(e.g., hormone refractory prostate adenocarcinoma), breast cancer, coloncancer, lung cancer (e.g. non-small cell lung cancer and small cell lungcancer), squamous cell head and neck cancer, urothelial cancer (e.g.bladder) and cancers with high microsatellite instability (MSI^(high)).Additionally, the disclosure includes refractory or recurrentmalignancies whose growth may be inhibited using the liposomes, or TLRprodrugs or co-formulated liposomes of the disclosure.

In additional embodiments, cancers that are treatable using theformulated and/or co-formulated liposomes or TLR prodrugs of the presentdisclosure include, but are not limited to, solid tumors (e.g., prostatecancer, colon cancer, esophageal cancer, endometrial cancer, ovariancancer, uterine cancer, renal cancer, hepatic cancer, pancreatic cancer,gastric cancer, breast cancer, lung cancer, cancers of the head andneck, thyroid cancer, glioblastoma, sarcoma, bladder cancer, etc.),hematological cancers (e.g., lymphoma, leukemia such as acutelymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chroniclymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), DLBCL,mantle cell lymphoma, Non-Hodgkin lymphoma (including relapsed orrefractory NHL and recurrent follicular), Hodgkin lymphoma or multiplemyeloma) and combinations of said cancers.

In further embodiments, cancers that are treatable using the formulatedand/or co-formulated liposomes or TLR prodrugs of the present disclosureinclude, but are not limited to, cholangiocarcinoma, bile duct cancer,triple negative breast cancer, rhabdomyosarcoma, small cell lung cancer,leiomyosarcoma, hepatocellular carcinoma, Ewing's sarcoma, brain cancer,brain tumor, astrocytoma, neuroblastoma, neurofibroma, basal cellcarcinoma, chondrosarcoma, epithelioid sarcoma, eye cancer, Fallopiantube cancer, gastrointestinal cancer, gastrointestinal stromal tumors,hairy cell leukemia, intestinal cancer, islet cell cancer, oral cancer,mouth cancer, throat cancer, laryngeal cancer, lip cancer, mesothelioma,neck cancer, nasal cavity cancer, ocular cancer, ocular melanoma, pelviccancer, rectal cancer, renal cell carcinoma, salivary gland cancer,sinus cancer, spinal cancer, tongue cancer, tubular carcinoma, urethralcancer, and ureteral cancer.

In addition, in some embodiments, the formulated and/or co-formulatedliposomes, or TLR prodrugs of the present disclosure can be used totreat sickle cell disease and sickle cell anemia.

Furthermore, in some embodiments, diseases and indications that aretreatable using the formulated and/or co-formulated liposomes, or TLRprodrugs of the present disclosure include, but are not limited tohematological cancers, sarcomas, lung cancers, gastrointestinal cancers,genitourinary tract cancers, liver cancers, bone cancers, nervous systemcancers, gynecological cancers, and skin cancers.

Exemplary hematological cancers include lymphomas and leukemias such asacute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML),acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL),chronic myelogenous leukemia (CML), diffuse large B-cell lymphoma(DLBCL), mantle cell lymphoma, Non-Hodgkin lymphoma (including relapsedor refractory NHL and recurrent follicular), Hodgkin lymphoma,myeloproliferative diseases (e.g., primary myelofibrosis (PMF),polycythemia vera (PV), and essential thrombocytosis (ET)),myelodysplasia syndrome (MDS), T-cell acute lymphoblastic lymphoma(T-ALL) and multiple myeloma (MM).

Exemplary sarcomas include chondrosarcoma, Ewing's sarcoma,osteosarcoma, rhabdomyosarcoma, angiosarcoma, fibrosarcoma, liposarcoma,myxoma, rhabdomyoma, rhabdosarcoma, fibroma, lipoma, harmatoma, andteratoma.

Exemplary lung cancers include non-small cell lung cancer (NSCLC), smallcell lung cancer, bronchogenic carcinoma (squamous cell,undifferentiated small cell, undifferentiated large cell,adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma,chondromatous hamartoma, and mesothelioma.

Exemplary gastrointestinal cancers include cancers of the esophagus(squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma),stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductaladenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors,vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors,Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma),large bowel (adenocarcinoma, tubular adenoma, villous adenoma,hamartoma, leiomyoma), and colorectal cancer.

Exemplary genitourinary tract cancers include cancers of the kidney(adenocarcinoma, Wilm's tumor [nephroblastoma]), bladder and urethra(squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma),prostate (adenocarcinoma, sarcoma), and testis (seminoma, teratoma,embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma,interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors,lipoma).

Exemplary liver cancers include hepatoma (hepatocellular carcinoma),cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellularadenoma, and hemangioma.

Exemplary bone cancers include, for example, osteogenic sarcoma(osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochronfroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma, and giant celltumors.

Exemplary nervous system cancers include cancers of the skull (osteoma,hemangioma, granuloma, xanthoma, osteitis deformans), meninges(meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma,meduoblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma,glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma,congenital tumors), and spinal cord (neurofibroma, meningioma, glioma,sarcoma), as well as neuroblastoma and Lhermitte-Duclos disease.

Exemplary gynecological cancers include cancers of the uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (ovarian carcinoma (serous cystadenocarcinoma,mucinous cystadenocarcinoma, unclassified carcinoma), granulosa-thecalcell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma),and fallopian tubes (carcinoma).

Exemplary skin cancers include melanoma, basal cell carcinoma, squamouscell carcinoma, Kaposi's sarcoma, moles dysplastic nevi, lipoma,angioma, dermatofibroma, and keloids. In some embodiments, diseases andindications that are treatable using the compounds of the presentdisclosure include, but are not limited to, sickle cell disease (e.g.;sickle cell anemia), triple-negative breast cancer (TNBC),myelodysplastic syndromes, testicular cancer, bile duct cancer,esophageal cancer, and urothelial carcinoma.

Additionally, TLR and/or kynurenine pathway blockade with formulatedand/or co-formulated liposomes, or TLR prodrugs of the presentdisclosure can also be used for treating infections such as viral,bacteria, fungus, and parasite infections.

The present disclosure provides a method for treating infections such asviral infections. The method includes administering to a patient, atherapeutically effective amount of a formulated and/or co-formulatedliposome or TLR prodrugs or any of the formulas as described herein(i.e., TR3, TR6, TR6(A), TR5, TR5(A) and/or TR5(B)) as recited in any ofthe claims and described herein, a salt thereof.

Examples of viruses causing infections treatable by methods of thepresent disclosure include, but are not limit to, human immunodeficiencyvirus, human papillomavirus, influenza, hepatitis A, B, C or D viruses,adenovirus, poxvirus, herpes simplex viruses, human cytomegalovirus,severe acute respiratory syndrome virus, Ebola virus, and measles virus.In some embodiments, viruses causing infections treatable by methods ofthe present disclosure include, but are not limit to, hepatitis (A, B,or C), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, EpsteinBarr virus), adenovirus, influenza virus, flaviviruses, echovirus,rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus,mumps virus, rotavirus, measles virus, rubella virus, parvovirus,vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscumvirus, poliovirus, rabies virus, JC virus and arboviral encephalitisvirus.

In addition, the present disclosure provides a method for treatingbacterial infections. The method includes administering to a patient, atherapeutically effective amount of a formulated and/or co-formulatedliposome or TLR prodrugs, or any of the formulas as described herein(i.e., TR3, TR6, TR6(A), TR5, TR5(A) and/or TR5(B)) as recited in any ofthe claims and described herein, or a salt thereof.

Examples of pathogenic bacteria causing infections treatable by methodsof the disclosure, include but are not limited to, chlamydia, rickettsiabacteria, mycobacteria, staphylococci, streptococci, pneumonococci,meningococci and conococci, klebsiella, proteus, serratia, pseudomonas,legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism,anthrax, plague, leptospirosis, and Lyme's disease bacteria.

In addition, the present disclosure provides a method for treatingfungus infections. The method includes administering to a patient, atherapeutically effective amount of a formulated and/or co-formulatedliposome or TLR prodrugs, or any of the formulas as described herein(i.e., TR3, TR6, TR6(A), TR5, TR5(A) and/or TR5(B)) as recited in any ofthe claims and described herein, or a salt thereof.

Examples of pathogenic fungi causing infections treatable by methods ofthe disclosure include, but are not limited to, Candida (albicans,krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans,Aspergillus (fumigatus, Niger, etc.), Genus Mucorales (Mucor, absidia,rhizophus), Sporothrix schenkii, Blastomyces dermatitidis,Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasmacapsulatum.

Additionally, the present disclosure provides a method for treatingparasite infections. The method includes administering to a patient, atherapeutically effective amount of a formulated and/or co-formulatedliposome or TLR prodrugs, or any of the formulas as described herein(i.e., TR3, TR6, TR6(A), TR5, TR5(A) and/or TR5(B)) as recited in any ofthe claims and described herein, or a salt thereof.

Examples of pathogenic parasites causing infections treatable by methodsof the disclosure include, but are not limited to, Entamoebahistolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp.,Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodiumvivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi,Leishmania donovani, Toxoplasma gondi, and Nippostrongylus brasiliensis.

In a further set of embodiments that are within the scope of thisdisclosure, the formulated and/or co-formulated liposomes, or TLRprodrugs, or any of the formulas as described herein (i.e., TR3, TR6,TR6(A), TR5, TR5(A) and/or TR5(B)) are useful in preventing or reducingthe risk of developing any of the diseases referred to in thisdisclosure; e.g., preventing or reducing the risk of developing adisease, condition or disorder in an individual who may be predisposedto the disease, condition or disorder but does not yet experience ordisplay the pathology or symptomatology of the disease.

XII.) Kits/Articles of Manufacture

For use in the laboratory, prognostic, prophylactic, diagnostic, andtherapeutic applications described herein, kits are within the scope ofthe invention. Such kits can comprise a carrier, package, or containerthat is compartmentalized to receive one or more containers such asvials, tubes, and the like, each of the container(s) comprising one ofthe separate elements to be used in the method, along with a label orinsert comprising instructions for use, such as a use described herein.For example, the container(s) can comprise a formulated and/orco-formulated liposome that is or can be detectably labeled and/or isloaded with a TLR prodrug of the disclosure. Kits can comprise acontainer comprising a drug unit. The kit can include all or part of theformulated and/or co-formulated liposomes and/or a TLR prodrug.

The kit of the invention will typically comprise the container describedabove, and one or more other containers associated therewith thatcomprise materials desirable from a commercial and user standpoint,including buffers, diluents, filters, needles, syringes; carrier,package, container, vial and/or tube labels listing contents and/orinstructions for use, and package inserts with instructions for use.

A label can be present on or with the container to indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, such as a prognostic, prophylactic, diagnostic, orlaboratory application, and can also indicate directions for either invivo or in vitro use, such as those described herein. Directions and orother information can also be included on an insert(s) or label(s) whichis included with or on the kit. The label can be on or associated withthe container. A label can be on a container when letters, numbers orother characters forming the label are molded or etched into thecontainer itself; a label can be associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. The label can indicate that the compositionis used for diagnosing, treating, prophylaxing or prognosing acondition, such as a cancer or other immunological disorder.

The terms “kit” and “article of manufacture” can be used as synonyms.

In another embodiment of the invention, an article(s) of manufacturecontaining compositions, such as formulated and/or co-formulatedliposomes and/or TLR prodrugs are within the scope of this disclosure.The article of manufacture typically comprises at least one containerand at least one label. Suitable containers include, for example,bottles, vials, syringes, and test tubes. The containers can be formedfrom a variety of materials such as glass, metal, or plastic. Thecontainer can hold formulated and/or co-formulated liposomes loaded withTLR prodrugs.

The container can alternatively hold a composition that is effective fortreating, diagnosis, prognosing or prophylaxing a condition and can havea sterile access port (for example the container can be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The active agents in the composition can beformulated and/or co-formulated liposomes loaded with TLR prodrugsand/or TLR prodrugs as disclosed herein.

The article of manufacture can further comprise a second containercomprising a pharmaceutically acceptable buffer, such asphosphate-buffered saline, Ringer's solution and/or dextrose solution.It can further include other materials desirable from a commercial anduser standpoint, including other buffers, diluents, filters, stirrers,needles, syringes, and/or package inserts with indications and/orinstructions for use.

Exemplary Embodiments

Among the provided embodiments are:

-   -   1) A TLR prodrug composition comprising,        -   (i) a drug moiety;        -   (ii) a lipid moiety; and        -   (iii) a linkage unit (“LU”),    -   whereby the drug moiety comprises a TLR agonist and whereby the        LU conjugates the drug moiety with the lipid moiety.    -   2) The TLR prodrug of claim 1, further comprising the chemical        structure set forth in FORMULA I.    -   3) The TLR prodrug of claim 1, further comprising the chemical        structure set forth in FORMULA II.    -   4) The TLR prodrug of claim 1, wherein the drug moiety comprises        the chemical structure set forth as TR5.    -   5) The TLR prodrug of claim 1, wherein the drug moiety comprises        the chemical structure set forth as TR5(A).    -   6) The TLR prodrug of claim 1, wherein the drug moiety comprises        the chemical structure set forth as TR6.    -   7) The TLR prodrug of claim 1, wherein the drug moiety comprises        the chemical structure set forth as TR6(A).    -   8) The TLR prodrug of claim 1, wherein the drug moiety comprises        the chemical structure set forth as TR3.    -   9) The TLR prodrug of claim 1, wherein the LU is a        hydromethylcarbamate linker.    -   10) The TLR prodrug of claim 1, wherein the lipid moiety        comprises a lipid set forth in Table I.    -   11) The TLR prodrug of claim 1, wherein the lipid moiety        comprises a lipid set forth in Table Ill.    -   12) The TLR prodrug of claim 1, wherein the lipid moiety        comprises CHEMS.    -   13) The TLR prodrug of claim 1, wherein the lipid moiety        comprises Stearic Acid.    -   14) A TLR prodrug composition comprising,        -   (i) a drug moiety, whereby the drug moiety comprises TR5;        -   (ii) a lipid moiety, whereby the lipid moiety comprises            CHEMS; and        -   (iii) LU, whereby the LU comprises a hydromethylcarbamate            linker.    -   15) A TLR prodrug composition comprising,        -   (i) a drug moiety, whereby the drug moiety comprises TR5;        -   (ii) a lipid moiety, whereby the lipid moiety comprises            Stearic Acid; and        -   (iii) a LU, whereby the LU comprises a hydromethylcarbamate            linker.    -   16) A TLR prodrug composition comprising,        -   (i) a drug moiety, whereby the drug moiety comprises TR5(A);        -   (ii) a lipid moiety, whereby the lipid moiety comprises            CHEMS; and        -   (iii) LU, whereby the LU comprises a hydromethylcarbamate            linker.    -   17) A TLR prodrug composition comprising,        -   (i) a drug moiety, whereby the drug moiety comprises TR5(A);        -   (ii) a lipid moiety, whereby the lipid moiety comprises            Stearic Acid; and        -   (iii) a LU, whereby the LU comprises a hydromethylcarbamate            linker.    -   18) A TLR prodrug composition comprising,        -   (i) a drug moiety, whereby the drug moiety comprises TR6;        -   (ii) a lipid moiety, whereby the lipid moiety comprises            CHEMS; and        -   (iii) LU, whereby the LU comprises a hydromethylcarbamate            linker.    -   19) A TLR prodrug composition comprising,        -   (i) a drug moiety, whereby the drug moiety comprises TR6;        -   (ii) a lipid moiety, whereby the lipid moiety comprises            Stearic Acid; and        -   (iii) a LU, whereby the LU comprises a hydromethylcarbamate            linker.    -   20) A TLR prodrug composition comprising,        -   (i) a drug moiety, whereby the drug moiety comprises TR6(A);        -   (ii) a lipid moiety, whereby the lipid moiety comprises            CHEMS; and        -   (iii) LU, whereby the LU comprises a hydromethylcarbamate            linker.    -   21) A TLR prodrug composition comprising,        -   (i) a drug moiety, whereby the drug moiety comprises TR6(A);        -   (ii) a lipid moiety, whereby the lipid moiety comprises            Stearic Acid; and        -   (iii) a LU, whereby the LU comprises a hydromethylcarbamate            linker.    -   22) A TLR prodrug composition comprising,        -   (i) a drug moiety, whereby the drug moiety comprises TR3;        -   (ii) a lipid moiety, whereby the lipid moiety comprises            Stearic Acid; and        -   (iii) a LU, whereby the LU comprises a hydromethylcarbamate            linker.    -   23) A liposome comprising, a TLR prodrug whereby the liposome        releases an active TLR inhibitor after cleavage of a LU.    -   24) The liposome of claim 23, wherein the LU is a        hydromethylcarbamate linker.    -   25) The liposome of claim 23, further comprising a helper lipid,        whereby the helper lipid is set forth in Table II.    -   26) The liposome of claim 23, wherein the TLR prodrug comprises        TR3, TRS, TR5(A), and/or TR5(B).    -   27) The liposome of claim 23, wherein the TLR prodrug comprises        TR6 and/or TR6(A).    -   28) The liposome of claim 23, whereby the liposome is further        co-formulated with an iNTK activator.    -   29) The liposome of claim 28, wherein the iNKT activator is        Alpha-galactosylceramide (α-GalCer).    -   30) The liposome of claim 23, whereby the liposome is further        co-formulated with an immune modulating agent, wherein the        immune modulating agent is selected from the group consisting of        other TLR agonists and /or prodrugs, immunogenic-cell death        inducing chemotherapeutics, IDO antagonists, STING agonists,        CTLA-4 inhibitors, PD-1/PD-L1 inhibitors and/or prodrugs        thereof.    -   31) The liposome of claim 23, whereby the liposome is further        co-formulated with an ICD-inducing chemotherapeutic, wherein the        ICD-inducing chemotherapeutic is selected from the group        consisting of DOX, MTO, OXA, CP, Bortezomib, Carfilzimib, or        Paclitaxel.    -   32) The liposome of claim 23, further comprising DOX.    -   33) The liposome of claim 23, further comprising MTO.    -   34) The liposome of claim 23, further comprising DOX.    -   35) The liposome of claim 23, further comprising MTO.    -   36) A kit comprising a liposome of claim 23.    -   37) A kit comprising a liposome of claim 26.    -   38) A kit comprising a liposome of claim 27.    -   39) A liposome comprising, a TLR lipid moiety, wherein the TLR        Lipid moiety comprises a chemical structure set forth in FORMULA        Ill.    -   40) A liposome comprising, a TLR lipid moiety, wherein the TLR        Lipid moiety comprises a chemical structure set forth in FORMULA        IV.    -   41) The liposome of claim 39, further comprising a helper lipid,        whereby the helper lipid is set forth in Table II.    -   42) The liposome of claim 39, further comprising a helper lipid,        whereby the helper lipid is set forth in Table II.    -   43) The liposome of claim 38, wherein the TLR prodrug comprises        TR8.    -   44) The liposome of claim 40, wherein the TLR prodrug comprises        TR11.    -   45) The liposome of claim 38, whereby the liposome is further        co-formulated with an iNTK activator.    -   46) The liposome of claim 45, wherein the iNKT activator is        Alpha-galactosylceramide (α-GalCer).    -   47) The liposome of claim 39, whereby the liposome is further        co-formulated with an iNTK activator.    -   48) The liposome of claim 47, wherein the iNKT activator is        Alpha-galactosylceramide (α-GalCer).    -   49) The liposome of claim 44, whereby the liposome is further        co-formulated with an iNTK activator.    -   50) The liposome of claim 49, wherein the iNKT activator is        Alpha-galactosylceramide (α-GalCer).    -   51) The liposome of claim 43, whereby the liposome is further        co-formulated with an iNTK activator.    -   52) The liposome of claim 51, wherein the iNKT activator is        Alpha-galactosylceramide (α-GalCer).    -   53) The liposome of claim 39, whereby the liposome is further        co-formulated with an immune modulating agent, wherein the        immune modulating agent is selected from the group consisting of        immunogenic-cell death inducing chemotherapeutics, IDO        antagonists, STING agonists, CTLA-4 inhibitors, PD-1/PD-L1        inhibitors and/or prodrugs thereof.    -   54) The liposome of claim 39, whereby the liposome is further        co-formulated with an ICD-inducing chemotherapeutic, wherein the        ICD-inducing chemotherapeutic is selected from the group        consisting of DOX, MTO, OXA, CP, Bortezomib, Carfilzimib, or        Paclitaxel.    -   55) The liposome of claim 40, whereby the liposome is further        co-formulated with an immune modulating agent, wherein the        immune modulating agent is selected from the group consisting of        immunogenic-cell death inducing chemotherapeutics, IDO        antagonists, STING agonists, CTLA-4 inhibitors, PD-1/PD-L1        inhibitors and/or prodrugs thereof.    -   56) The liposome of claim 40, whereby the liposome is further        co-formulated with an ICD-inducing chemotherapeutic, wherein the        ICD-inducing chemotherapeutic is selected from the group        consisting of DOX, MTO, OXA, CP, Bortezomib, Carfilzimib, or        Paclitaxel.    -   57) A method of treating a subject suffering or diagnosed with        cancer comprising,        -   (i) administering to a subject in need of such treatment an            effective amount of a liposome, wherein the liposome            comprises a TLR prodrug; and        -   (ii) a pharmaceutically acceptable salt thereof.    -   58) The method of claim 57, wherein the TLR prodrug comprises        TR3, TR5, TR5(A), and/or TR5(B).    -   59) The method of claim 57, wherein the liposome comprises TR3,        TR5, TR5(A), and/or TR5(B) further co-formulated with and        ICD-inducing chemotherapeutic.    -   60) The method of claim 57, wherein the liposome comprises TR3,        TR5, TR5(A), and/or TR5(B) further co-formulated with an immune        modulating agent.    -   61) The method of claim 57, wherein the liposome comprises TR3,        TR5, TR5(A), and/or TR5(B) further co-formulated with an iNTK        activator.    -   62) The method of claim 58, wherein the iNTK activator is        Alpha-galactosylceramide (α-GalCer).    -   63) A method of treating a subject suffering or diagnosed with        cancer comprising,        -   (i) administering to a subject in need of such treatment an            effective amount of a liposome, wherein the liposome            comprises a TLR prodrug; and        -   (ii) a pharmaceutically acceptable salt thereof.    -   64) The method of claim 63, wherein the TLR prodrug comprises        TR6 and/or TR6(A).    -   65) The method of claim 63, wherein the liposome comprises TR6        and/or TR6(A) further co-formulated with and ICD-inducing        chemotherapeutic.    -   66) The method of claim 63, wherein the liposome comprises TR6        and/or TR6(A) further co-formulated with an immune modulating        agent.    -   67) The method of claim 63, wherein the liposome comprises TR6        and/or TR6(A) further co-formulated with an iNTK activator.    -   68) The method of claim 67, wherein the iNTK activator is        Alpha-galactosylceramide (α-GalCer).    -   69) A method of treating a subject suffering or diagnosed with        cancer comprising,        -   (i) administering to a subject in need of such treatment an            effective amount of a liposome, wherein the liposome            comprises a TLR lipid moiety; and        -   (ii) a pharmaceutically acceptable salt thereof.    -   70) The method of claim 69, wherein the TLR lipid moiety        comprises TR8.    -   71) The method of claim 69, wherein the liposome comprises TR8        further co-formulated with and ICD-inducing chemotherapeutic.    -   72) The method of claim 69, wherein the liposome comprises TR8        further co-formulated with an immune modulating agent.    -   73) The method of claim 69, wherein the liposome comprises TR8        further co-formulated with an iNTK activator.    -   74) The method of claim 73, wherein the iNTK activator is        Alpha-galactosylceramide (α-GalCer).    -   75) The method of claim 69, wherein the liposome comprises TR8        further co-formulated with ID3.    -   76) A method of treating a subject suffering or diagnosed with        cancer comprising,        -   (i) administering to a subject in need of such treatment an            effective amount of a liposome, wherein the liposome            comprises a TLR lipid moiety; and        -   (ii) a pharmaceutically acceptable salt thereof.    -   77) The method of claim 76, wherein the TLR lipid moiety        comprises TR11.    -   78) The method of claim 76, wherein the liposome comprises TR11        further co-formulated with and ICD-inducing chemotherapeutic.    -   79) The method of claim 76, wherein the liposome comprises TR11        further co-formulated with an immune modulating agent.    -   80) The method of claim 76, wherein the liposome comprises TR11        further co-formulated with an iNTK activator.    -   81) The method of claim 80, wherein the iNTK activator is        Alpha-galactosylceramide (α-GalCer).

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples that follow, none of which is intended tolimit the scope of the invention.

Example 1: Chemical Synthesis of TR5(B) Prodrug Comprising VariousLipids

Chemical synthesis of a TR5(B) prodrug comprising various lipids, setforth for example, in Table(s) I and III is synthesized using thefollowing protocol. First, Quinoline (1) is treated with valericanhydride to yield Intermediate 2. Then, 4-aminobutanol (3) is treatedwith benzylchloroformate (4) to yield intermediate Intermediate 5. Then,Intermediate 5 is treated with N-hydroxyphthalimide, triphenylphosphine,and DIAD, followed by hydrazine to yield Intermediate 6. Then,Intermediate 6 and Intermediate 2 were heated with triethylamine toyield Intermediate 7. Then, Intermediate 7 was treated sequentially withmCPBA, ammonium hydroxide, benzenesulfonyl chloride, then heated withHCl to yield Intermediate 8. Finally, Intermediate 8 was treated withvarious lipid carboxylic acids to yield final product denoted TR5(B)(FIG. 1 ).

Example 2: Chemical Synthesis of TR6 Prodrug Comprising Various Lipids

Chemical synthesis of a TR6 prodrug comprising various lipids, set forthfor example, in Table(s) I and III is synthesized using the followingprotocol. First, Fluoronitroaniline (1) is treated with methyl amine toyield Compound 2. Then, Compound 2 was treated withtrimethylorthoformate to yield benzimidazole (3). Then, Benzimidazole(3) was treated with Compound 4 to yield Compound 5. Then, Compound 5was treated with ammonium acetate to yield Compound 6. Then, Compound 6was treated with chloromethylchloroformate (7) to yield Compound 8.Finally, Compound 8 was treated with various lipid acids (9) to yieldfinal product denoted TR6 (FIG. 2 ).

Example 3: Chemical Synthesis of TR6 Prodrug Intermediate

In another experiment, a chemical synthesis of the TR6 prodrugintermediate was performed in the following manner. To a solution ofCu-T12.9 (5.50 g, 15.1 mmol, 1.00 eq) in THF (75.0 mL) was added LiHMDS(1 M, 18.2 mL, 1.20 eq) at −70° C. and the reaction mixture was stirredat −70° C. for 0.5 hr. Then a solution of compound 2a (2.94 g, 22.7mmol, 2.02 mL, 1.50 eq) in THF (10 mL) was added to the mixture at −70°C. and stirred at −70° C. for another 1.5 hrs. LCMS showed that Cu-T12.9(RT=0.934 min) was consumed incompletely, and the desired mass (RT=0.966min) was detected. The reaction mixture was poured into saturated citricacid solution (100 mL) and extracted with ethyl acetate (150 mL*3). Theorganic layer was washed with brine (150 mL*2), dried over Na₂SO₄,filtered, and concentrated to give the crude product confirmed by LCMSand HPLC). The crude product was purified by prep-HPLC (column:Phenomenex luna C18 250*80 mm*10 um; mobile phase: [water (0.1%TFA)-ACN]; B %: 38 ACN %-68 ACN %, 21 min) and extracted with ethylacetate (200 mL*2) directly. The combined organic layer was washed withbrine (200 mL*3), dried over Na₂SO₄, filtered, and concentrated to givecompound 2 (2.50 g, 5.50 mmol, 36.2% yield) as a yellow solid. Theresulting compounds are shown in FIG. 6 .

Example 4: Chemical Synthesis of TR6 Prodrug Comprising CholesterolHemisuccinate (“CHEMS”)

In another experiment, an TR6 Prodrug comprising cholesterolhemisuccinate (“CHEMS”) was synthesized in the following manner.Briefly, a solution of cholesterol hemisuccinate (2.68 g, 5.50 mmol,1.00 eq) in DMF (150 mL) was added Ag₂CO₃ (2.27 g, 8.25 mmol, 374 uL,1.50 eq) at 25° C. The reaction mixture was stirred at 25° C. for 0.5hr. Then compound 2 (2.50 g, 5.50 mmol, 1.00 eq) and Nal (1.24 g, 8.25mmol, 1.50 eq) were added to the mixture. After addition, the reactionmixture was stirred at 80° C. for 12 hrs. LCMS showed that the reactionwas completed, and the desired mass (RT=1.465 mins) was detected. Thereaction mixture was cooled to 25° C. and filtered through a pad ofcelite and washed with DMF (200 mL). The filtrate was concentrated at50° C. to give the crude product, which was purified by reverse-MPLC(TFA condition) and then concentrated under reduced pressure to give thecrude product. The crude product was combined with EW15710-150-P1 (˜700mg) to purified by column chromatography (SiO₂, Petroleum ether: Ethylacetate=5:1 to 2:1, R_(f)=0.6), which was detected by TLC (Petroleumether: Ethyl acetate=1:1, R_(f)=0.6, PMA). Target 1 (1.80 g, 1.97 mmol,29.8% yield, 99.0% purity) was obtained as a yellow solid, which wasconfirmed by ¹H NMR, ¹⁹F NMR, LCMS, HPLC, SFC, and DSC. The resultingcompound(s) are shown in FIG. 7 .

Example 5: Chemical Synthesis of TR3 Prodrug Comprising Stearic Acid

In another experiment, a TR3 Prodrug comprising stearic acid wassynthesized in the following manner. Briefly, to a solution of compound4 (9.86 g, 15.9 mmol, 94.7% purity, 1.00 eq) in CHCl₃ (100 mL) was addedm-CPBA (4.86 g, 23.9 mmol, 85.0% purity, 1.50 eq) in portions. Then, themixture was stirred at 80° C. for five (5) hrs. LCMS showed 6.01% ofcompound 4 (Retention Time=1.041 mins) was remained and desired mass(Retention Time=1.149 mins) was detected. Then NH₃.H₂O (107 g, 920 mmol,118 mL, 30% purity, 57.6 eq) was added and the mixture was stirred at15° C. for thirty (30) mins. Finally, TosCl (3.65 g, 19.1 mmol, 1.20 eq)was added in one portion. The mixture was stirred at 15° C. for twelve(12) hrs. LCMS showed that compound 4 was consumed and the desired mass(RT=1.049 mins) was detected. The reaction mixture was separated byseparating funnel. The aqueous phase was extracted with DCM (100 mL*2).The combined organic phase was washed with brine (100 mL), dried overNa₂SO₄, filtered, and concentrated under vacuum. The crude productcombination was purified by prep-HPLC (column: Welch Ultimate XB-SiOH250*50*10 um; mobile phase: [Hexane-EtOH (0.1% NH₃ H₂O)]; B %: 1%-40%,20 min) and further purified by MPLC (SiO₂, Dichloromethane/Methanol=1/0to 100/1, Dichloromethane/Methanol=10/1, R_(f)=0.35). The Target A(485.61 mg, 767 μmol, 4.33% yield, 94.8% purity) was obtained asoff-white solid, which was confirmed by ¹H NMR, LCMS, and HPLC. Theresulting compound(s) are shown in FIG. 8 .

Example 6: Synthesis and Characterization of LNP-TR6 Liposome

A liposome comprising the TR6 prodrug (denoted LNP-TR6) was synthesizedand characterized using a microfluidics technique performed on aNanoAssemblr Bench top instrument (Precision NanoSystems) in thefollowing manner. Briefly, a stock solution of each lipid component;Hydro Soy PC[(HSPC: L-α-phosphatidylcholine, hydrogenated (Soy)],Cholesterol, DSPE-PEG(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000], and TR6 were prepared in ethanol at the concentration of20 mg/ml. Each of the lipid components (HSPC, CHOL, DSPE-PEG, and TR6)were mixed together in a molar ratio of 40:31:26:3 to synthesizeLNP-TR6. The size of the LNP-TR6 depend on various parameter(s) such asflow rate, temperature, and concentration of the lipid mixture. Theoptimized ratio of the lipid mixture at the molar ratio of 40:31:26:3was preheated at 50° C. using the heating block attachment in themicrofluidizer. The aqueous phase containing 1 mM PBS buffer was alsopreheated at 50° C. before passing through the microfluidics cartridgeat the flow rate of 3:1 (aqueous: organic phase, lipid mixture). Thesolvent was removed using dialysis membrane of cut off 12 KDa size(Sigma Aldrich) against DI water for at least twenty-four (24) hrs. TheDI water was changed at least five (5) times during the period oftwenty-four (24) hrs. to maximize the removal of the solvent. After theremoval of the solvent, the LNP-TR6 was concentrated according to theneed using Amicon centrifugal filtration device (cut off size 10 KDa, at3000 g).

Characterization of the LNP-TR6 liposome was determined using a MalvernZetasizer (Malvern Instrumentation Co., Westborough, Mass., USA). Two(2) ml of LNP-TR6 liposomes (concentration of the liposome was of 0.5-1mg/ml) was placed in a 4-sided, clear, plastic cuvette and analyzeddirectly at 25° C. The results shown in FIG. 9 show the Zav size of thenanoparticles were approximately 83 nm with a PDI of approximately0.164.

Additionally, Zeta potential of the LNP-TR6 liposomes in aqueousdispersion was determined using a Malvern zeta seizer Instrument (MalvemInstrumentation Co, Westborough, Mass., USA). Briefly, approximately one(1) ml of the liposome (concentration approximately 2 mg/ml in 20 mMNaCl) was placed in a disposable capillary zeta potential cell availablefor the Zetasizer. The measurement was done at 25° C. The results showthe Zeta potential determination of LNP-TR6 was approximately −16.2 mV(FIG. 10 ).

Example 7: Synthesis and Characterization of LNP-TR5 Liposome

In another experiment, a liposome comprising the TR5 prodrug (denotedLNP-TR5) was synthesized and characterized using a microfluidicstechnique performed on a NanoAssemblr Bench top instrument (PrecisionNanoSystems) in the following manner. Briefly, a stock solution of eachlipid component; Hydro Soy PC[(HSPC: L-α-phosphatidylcholine,hydrogenated (Soy)], Cholesterol, DSPE-PEG(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000], and TR5 were prepared in ethanol at the concentration of10 mg/ml. Each of the lipid components (HSPC, CHOL, DSPE-PEG, and TR5)were mixed together in a molar ratio of 53:33:9:5 to synthesize LNP-TR5.The size of the LNP-TR5 depend on various parameter(s) such as flowrate, temperature, and concentration of the lipid mixture. The optimizedratio of the lipid mixture at the molar ratio of 53:33:9:5 was preheatedat 45° C. using the heating block attachment in the microfluidizer. Theaqueous phase containing 1 mM PBS buffer was also preheated at 45° C.before passing through the microfluidics cartridge at the flow rate of3.5:1 (aqueous: organic phase, lipid mixture). The solvent was removedusing dialysis membrane of cut off 12 KDa size (Sigma Aldrich) againstDI water for at least twenty-four (24) hrs. The DI water was changed atleast five (5) times during the period of twenty-four (24) hrs. tomaximize the removal of the solvent. After the removal of the solvent,the LNP-TR6 was concentrated according to the need using Amiconcentrifugal filtration device (cut off size 10 KDa, at 3000 g).

Characterization of the LNP-TR5 liposome was determined using a MalvernZetasizer (Malvern Instrumentation Co., Westborough, Mass., USA). Two(2) ml of LNP-TR5 liposomes (concentration of the liposome was of 0.5-1mg/ml) was placed in a 4-sided, clear, plastic cuvette and analyzeddirectly at 25° C. The results shown in FIG. 11 show the Zav size of thenanoparticles were approximately 85 nm with a PDI of approximately0.096.

Additionally, Zeta potential of the LNP-TR5 liposomes in aqueousdispersion was determined using a Malvern zeta seizer Instrument(Malvern Instrumentation Co, Westborough, Mass., USA). Briefly,approximately one (1) ml of the liposome (concentration approximately 2mg/ml in 20 mM NaCl) was placed in a disposable capillary zeta potentialcell available for the Zetasizer. The measurement was done at 25° C. Theresults show the Zeta potential determination of LNP-TR5 wasapproximately −13.1 mV (FIG. 12 ).

Example 8: Synthesis and Characterization of LNP-TR3 Liposome

In another experiment, a liposome comprising the TR3 prodrug (denotedLNP-TR3) was synthesized and characterized using a microfluidicstechnique performed on a NanoAssemblr Bench top instrument (PrecisionNanoSystems) in the following manner. Briefly, a stock solution of eachlipid component; HSPC, Cholesterol, DSPE-PEG was prepared in ethanol ata concentration of 20 mg/ml. The TR3 prodrug was prepared in ethanol atthe concentration of 2 mg/ml. Each of the lipid components (HSPC, CHOL,DSPE-PEG, and TR3) were mixed together in a molar ratio of 53.5:33.5:6:5and then diluted with ethanol to obtain a lipid concentration ofapproximately 9 mg/ml to synthesize LNP-TR3. The size of the LNP-TR3depend on various parameter(s) such as flow rate, temperature, andconcentration of the lipid mixture. The optimized ratio of the lipidmixture at the molar ratio of 53.5:33.5:6:5 was preheated at 45° C.using the heating block attachment in the microfluidizer. The aqueousphase containing 1 mM PBS buffer was also preheated at 45° C. beforepassing through the microfluidics cartridge at the flow rate of 3.5:1(aqueous: organic phase, lipid mixture). The solvent was removed usingdialysis membrane of cut off 12 KDa size (Sigma Aldrich) against DIwater for at least twenty-four (24) hrs. The DI water was changed atleast five (5) times during the period of twenty-four (24) hrs. tomaximize the removal of the solvent. After the removal of the solvent,the LNP-TR3 was concentrated according to the need using Amiconcentrifugal filtration device (cut off size 10 KDa, at 3000 g).

Characterization of the LNP-TR3 liposome was determined using a MalvernZetasizer (Malvern Instrumentation Co., Westborough, Mass., USA). Two(2) ml of LNP-TR3 liposomes (concentration of the liposome was of 0.5-1mg/ml) was placed in a 4-sided, clear, plastic cuvette and analyzeddirectly at 25° C. The results shown in FIG. 13 show the Zav size of thenanoparticles were approximately 88 nm with a PDI of approximately0.125.

Additionally, Zeta potential of the LNP-TR3 liposomes in aqueousdispersion was determined using a Malvern zeta seizer Instrument(Malvern Instrumentation Co, Westborough, Mass., USA). Briefly,approximately one (1) ml of the liposome (concentration approximately 2mg/ml in 20 mM NaCl) was placed in a disposable capillary zeta potentialcell available for the Zetasizer. The measurement was done at 25° C. Theresults show the Zeta potential determination of LNP-TR3 wasapproximately −13.2 mV (FIG. 14 ).

Example 9: Synthesis and Characterization of LNP-TR8 Liposome

In another experiment, a liposome comprising the TR8 prodrug (denotedLNP-TR8) was synthesized and characterized using a microfluidicstechnique performed on a NanoAssemblr Bench top instrument (PrecisionNanoSystems) in the following manner. Briefly, a stock solution of eachlipid component; HSPC, Cholesterol, DSPE-PEG was prepared separately.MPLA (PHAD®) Monophosphoryl Lipid A (Synthetic) (PHAD) stock solutionwas prepared in dimethyl sulfoxide (10 mg/ml). Each of the lipidcomponents (HSPC, CHOL, DSPE-PEG, and MPLA) were mixed together in amolar ratio of 55:37.5:3 and then diluted with ethanol to obtain a lipidconcentration of approximately 10 mg/ml. The size of the LNP-TR8 dependon various parameter(s) such as flow rate, temperature, andconcentration of the lipid mixture. The optimized ratio of the lipidmixture at the molar ratio of 55:37:5:3 was preheated at 45° C. usingthe heating block attachment in the microfluidizer. The aqueous phasecontaining 1 mM PBS buffer was also preheated at 45° C. before passingthrough the microfluidics cartridge at the flow rate of 5:1 (aqueous:organic phase, lipid mixture). The solvent was removed using dialysismembrane of cut off 12 KDa size (Sigma Aldrich) against DI water for atleast twenty-four (24) hrs. The DI water was changed at least five (5)times during the period of twenty-four (24) hrs. to maximize the removalof the solvent. After the removal of the solvent, the LNP-TR8 wasconcentrated according to the need using Amicon centrifugal filtrationdevice (cut off size 10 KDa, at 3000 g).

Characterization of the LNP-TR8 liposome was determined using a MalvernZetasizer (Malvern Instrumentation Co., Westborough, Mass., USA). Two(2) ml of LNP-TR8 liposomes (concentration of the liposome was of 0.5-1mg/ml) was placed in a 4-sided, clear, plastic cuvette and analyzeddirectly at 25° C. The results shown in FIG. 15 show the Zav size of thenanoparticles were approximately 89 nm with a PDI of approximately0.240.

Additionally, Zeta potential of the LNP-TR8 liposomes in aqueousdispersion was determined using a Malvern zeta seizer Instrument(Malvern Instrumentation Co, Westborough, Mass., USA). Briefly,approximately one (1) ml of the liposome (concentration approximately 2mg/ml in 20 mM NaCl) was placed in a disposable capillary zeta potentialcell available for the Zetasizer. The measurement was done at 25° C. Theresults show the Zeta potential determination of LNP-TR8 wasapproximately −9.8 mV (FIG. 16 ).

Example 10: Synthesis and Characterization of LNP-ID3-TR8 Liposome

In another experiment, a liposome comprising the ID3 prodrug and the TR8prodrug (denoted LNP-ID3-TR8) was synthesized and characterized using amicrofluidics technique performed on a NanoAssemblr Bench top instrument(Precision NanoSystems) in the following manner. Briefly, a stocksolution of each lipid component; HSPC, Cholesterol, DSPE-PEG wasprepared in ethanol at a concentration of 20 mg/ml. Meanwhile, a stocksolution of ID3 prodrug (20 mg/ml) was prepared in acetonitrile, while astock solution of MPLA (10 mg/ml) was prepared in DSMO. Each of thelipid components (HSPC, CHOL, DSPE-PEG, ID3, and MPLA) were mixedtogether in a molar ratio of 54:28:12:3:3 and then diluted with ethanolto obtain a lipid concentration of approximately 10 mg/ml. The size ofthe LNP-ID3-TR8 depend on various parameter(s) such as flow rate,temperature, and concentration of the lipid mixture. The optimized ratioof the lipid mixture at the molar ratio of 54:28:12:3:3 was preheated at45° C. using the heating block attachment in the microfluidizer. Theaqueous phase containing 1 mM PBS buffer was also preheated at 45° C.before passing through the microfluidics cartridge at the flow rate of3:1 (aqueous: organic phase, lipid mixture). Accordingly, the liposomeformulation was synthesized in such a way so that the molar ratio ofID3:TR8 remain at a 4:1 ratio. The solvent was removed using dialysismembrane of cut off 12 KDa size (Sigma Aldrich) against DI water for atleast twenty-four (24) hrs. The DI water was changed at least five (5)times during the period of twenty-four (24) hrs. to maximize the removalof the solvent. After the removal of the solvent, the LNP-TR8 wasconcentrated according to the need using Amicon centrifugal filtrationdevice (cut off size 10 KDa, at 3000 g).

Characterization of the LNP-ID3-TR8 liposome was determined using aMalvem Zetasizer (Malvem Instrumentation Co., Westborough, Mass., USA).Two (2) ml of LNP-ID3-TR8 liposomes (concentration of the liposome wasof 0.5-1 mg/ml) was placed in a 4-sided, clear, plastic cuvette andanalyzed directly at 25° C. The results shown in FIG. 17 show the Zavsize of the nanoparticles were approximately 82 nm with a PDI ofapproximately 0.170.

Additionally, Zeta potential of the LNP-ID3-TR8 liposomes in aqueousdispersion was determined using a Malvern zeta seizer Instrument (MalvemInstrumentation Co, Westborough, Mass., USA). Briefly, approximately one(1) ml of the liposome (concentration approximately 2 mg/ml in 20 mMNaCl) was placed in a disposable capillary zeta potential cell availablefor the Zetasizer. The measurement was done at 25° C. The results showthe Zeta potential determination of LNP-ID3-TR8 was approximately −11.9mV (FIG. 18 ).

Example 11: Tumor Inhibition of LNP-TR5 in Combination with LNP-DOXUsing B16F10 Cells In Vivo

Evaluation of LNP-TR5 in combination with LNP-DOX was performed usingthe following protocols. Briefly, murine melanoma cancer B16F10 cells(0.2×10⁶) were inoculated subcutaneously in the right rear flank regionof C57BL/6 mice. Animals were treated with vehicle control, LNP-DOX(Doxorubicin in liposome form) at 3 mg/kg, and combination of LNP-DOX at3 mg/kg and LNP-TR5 at 1 mg/kg, two times weekly through i.v injection.Tumor volumes were measured three (3) times in two dimensions using acaliper, and the volume was calculated using the formula: V=(L×W×W)×0.5,where V is tumor volume, L is tumor length (the longest tumordimension), and W is tumor width (the longest tumor dimensionperpendicular to L). The tumor growth inhibition (TGI) was calculatedbased on the tumor size data of day twenty (20).

The results show treatment with LNP-DOX alone produces a significanttumor growth inhibition. The TGI was calculated at 66.37% (p<0.01) whencompared with the vehicle treated group. Additionally, combinationLNP-TR5 and LNP-DOX also produced significant anti-tumor activity. TheTGI was calculated at 52.73% (p<0.05). (See, FIG. 19 ).

Example 12: Tumor Inhibition of LNP-TR6 in Combination with LNP-NK1 andLNP-TR6 in Combination with LNP-TR8 and LNP-MTO Using B16F10 Cells InVivo

Evaluation of LNP-TR6 in combination with LNP-NK1 and LNP-TR6 wasperformed using the following protocols. Briefly, murine melanoma cancerB16F10 cells (0.2×10⁶) were inoculated subcutaneously in the right rearflank region of C57BU6 mice. Animals were treated with vehicle control,LNP-MTO (Mitoxantrone dihydrochloride in liposome form) at 2 mg/kg,combination of LNP-TR6 at 3 mg/kg and LNP-NK1 (KRN7000 in liposome form)at 0.03 mg/kg, and combination of LNP-TR6 at 2 mg/kg, LNP-TR8 at 0.6mg/kg and LNP-MTO at 2 mg/kg, two times weekly through i.v injection.Tumor volumes were measured three (3) times in two dimensions using acaliper, and the volume was calculated using the formula: V=(L×W×W)×0.5,where V is tumor volume, L is tumor length (the longest tumordimension), and W is tumor width (the longest tumor dimensionperpendicular to L). The tumor growth inhibition (TGI) was calculatedbased on the tumor size data of day eighteen (18).

The results show that treatment with LNP-TR6+LNP-NK1 andLNP-TR6+LNP-TR8+LNP-MTO produce a significant tumor growth inhibition,when compared to vehicle treated group. The TGIs were calculated at49.87% and 57.02%, respectively (p<0.01). (See, FIG. 20 ).

Example 13: Tumor Inhibition of LNP-TR5 and LNP-TR6 in Combination withLNP-AR5 Using B16F10 Cells In Vivo

Evaluation of LNP-TR6 in combination with LNP-AR5 and LNP-TR5 incombination with LNP-AR5 was performed using the following protocols.Briefly, murine melanoma cancer B16F10 cells (0.2×10⁶) were inoculatedsubcutaneously in the right rear flank region of C57BU6 mice. Animalswere treated with vehicle control, LNP-AR5 (ZM241385-Stearic Acid inliposome form) at 3 mg/kg, combination of LNP-AR5 and LNP-TR6 at 3 mg/kgand combination of LNP-AR5 and LNP-TR5 at 3 mg/kg two times weeklythrough i.v injection. Tumor volumes were measured three (3) times intwo dimensions using a caliper, and the volume was calculated using theformula: V=(L×W×W)×0.5, where V is tumor volume, L is tumor length (thelongest tumor dimension), and W is tumor width (the longest tumordimension perpendicular to L). The tumor growth inhibition (TGI) wascalculated based on the tumor size data of day fifteen (15).

The results show treatment with LNP-AR5+LNP-TR5 produces significantanti-tumor activities. The TGI was calculated at 30.72% (all p<0.05)when compared to vehicle treated group. Insignificant anti-tumoractivities were observed in LNP-AR5 and LNP-AR5+LNP-TR6 groups. TGIswere calculated at 14.86% and 21.52%, respectively. (See, FIG. 21 ).

Example 14: Tumor Inhibition of LNP-TR5 in Combination with LNP-AR5 andLNP-DOX Using EMT6 Cells In Vivo

Evaluation of LNP-TR5 in combination with LNP-AR5 and LNP-DOX wasperformed using the following protocols. Briefly, murine breast cancerEMT6 cells (0.1×10⁶) were inoculated subcutaneously in the right rearflank region of Balb/c mice. Animals were treated with vehicle control,Doxorubicin (LNP-DOX) at 3 mg/kg, and combination ofLNP-DOX+LNP-AR5+LNP-TR5 at 3 mg/kg through i.v injection. Afterreceiving two (2) doses of LNP-DOX, the LNP-DOX treatment was replacedwith vehicle. Tumor volumes were measured three (3) times in twodimensions using a caliper, and the volume was calculated using theformula: V=(L×W×W)×0.5, where V is tumor volume, L is tumor length (thelongest tumor dimension), and W is tumor width (the longest tumordimension perpendicular to L). The tumor growth inhibition (TGI) wascalculated based on the tumor size data of day twenty-one (21).

The results show, treatment of LNP-DOX alone at 3 mg/kg for two (2)doses in one (1) week produces significant anti-tumor activity. The TGIwas calculated at 68.74% (p<0.05) when compared to vehicle treatedgroup. Additionally, improved anti-tumor activities were observed whencombining the LNP-DOX treatment with LNP-AR5+LNP-TR5. The TGI was at87.92%, (all p<0.05). (See, FIG. 22 ).

Example 15: Tumor Inhibition Studies of LNP-TR5 and LNP-TR6 in MultipleCombination(s) Using H22 Cells In Vivo

Evaluation of LNP-TR5 and LNP-TR6 in a plurality of combination(s) wasperformed using the following protocols. Briefly, hepatocellularcarcinoma H22 cells (1×10⁶) were inoculated subcutaneously in the rightrear flank region of Balb/c mice. Animals were treated with vehiclecontrol, anti-PD1 antibody at 10 mg/kg, combination of LNP-AR5 at 4mg/kg and LNP-TR5 at 4 mg/kg for the first two (2) doses and 2 mg/kg forthe remainder of the study, combination of LNP-AR5 and LNP-TR6 at 4mg/kg, combination of anti-PD1 antibody at 10 mg/kg, LNP-AR5 at 4 mg/kgand LNP-TR5 at 4 mg/kg for the first two (2) doses and 2 mg/kg for theremainder of the study, and combination of LNP-ID3, LNP-AR5 at 3 mg/kgfor first two (2) doses and 3.5 mg/kg for the remainder of the study,and LNP-TR5 at 3 mg/kg for the first two (2) doses and 2 mg/kg for theremainder of the study through i.v. injection. Tumor volumes weremeasured 3 times in two dimensions using a caliper, and the volume wascalculated using the formula: V=(L×W×W)×0.5, where V is tumor volume, Lis tumor length (the longest tumor dimension) and W is tumor width (thelongest tumor dimension perpendicular to L). The tumor growth inhibition(TGI) was calculated based on the tumor size data of day fourteen (14).

The results show that treatment with Anti-PD-1+LNP-AR5+LNP-TR5 producessignificant anti-tumor activities. The TGI was calculated at 74.98% (allp<0.05) when compared to vehicle treated group. Insignificant anti-tumoractivity was observed in LNP-AR5+LNP-TR5, and LNP-ID3+LNP-AR5+LNP-TR5.The TGIs were calculated at 55.8%, and 48.83%, respectively. (See, FIG.23 ).

Example 16: Tumor Inhibition Studies of LNP-TR5 and LNP-TR6 in MultipleCombination(s) in Colorectal Cancer Cells In Vivo

Evaluation of LNP-TR5 and LNP-TR6 in a plurality of combination(s) wasperformed using the following protocols. Briefly, colorectal cancercells (1×10⁶) were inoculated subcutaneously in the right rear flankregion of C57BU6 mice. Animals were treated with vehicle control,Doxorubicin (LNP-DOX) at 4 mg/kg, anti-PD1 antibody at 10 mg/kg,combination of LNP-AR5 at 4 mg/kg and LNP-TR5 at 4 mg/kg for the firsttwo (2) doses and 2 mg/kg for the remainder of the study, a combinationof LNP-AR5 and LNP-TR6 at 4 mg/kg, a combination of LNP-DOX, LNP-AR5 andLNP-TR6 at 4 mg/kg, a combination of LNP-DOX, LNP-AR5 at 4 mg/kg andLNP-TR5 at 4 mg/kg for the first two (2) doses and 2 mg/kg for theremainder of the study, a combination of anti-PD1 antibody at 10 mg/kg,LNP-AR5 at 4 mg/kg and LNP-TR5 at 4 mg/kg for the first two (2) dosesand 2 mg/kg for the remainder of the study, and a combination of LNP-ID3and LNP-AR5 at 3 mg/kg for first two (2) doses and 3.5 mg/kg for theremainder of the study, and LNP-TR5 at 3 mg/kg for the first two (2)doses and 2 mg/kg for the remainder of the study through i.v. injection.

The study further included a dosing regimen wherein the groups receivinga dose of LNP-DOX, the first doses included solely LNP-DOX andsubsequently the LNP-DOX was halted and mice were dosed with otherdrugs.

Tumor volumes were measured three (3) times in two dimensions using acaliper, and the volume was calculated using the formula: V=(L×W×W)×0.5,where V is tumor volume, L is tumor length (the longest tumor dimension)and W is tumor width (the longest tumor dimension perpendicular to L).The tumor growth inhibition (TGI) was calculated based on the tumor sizedata of day nineteen (19).

The results show that treatment of LNP-DOX alone at 4 mg/kg producessignificant anti-tumor activity. The TGI was calculated at 74.39%(p<0.05) when compared to vehicle treated group. Additionally,combination treatment with LNP-AR5+LNP-TR5+LNP-DOX followed byLNP-AR5+LNP-TR6+LNP-DOX followed byLNP-AR5+LNP-TR5+Anti-PD-1+LNP-AR5+LNP-TR5, and LNP-ID3+LNP-AR5+LNP-TR5also produce significant anti-tumor activities. The TGIs were calculatedat 77.35%, 90.31%, 87.67%, 91.33% and 73.77% (all p<0.05), respectively.(See, FIG. 24 ).

Example 17: In Vitro Validation of TR6 Prodrug in Liposome formMechanism of Action

To confirm in vitro biological activity of LNP-TR6 and to furtherconfirm targeting of TLR1/2, the following experiment(s) were performedusing a RAW-Blue™ Cells and QUANTI-Blue™ assay (InvivoGen, San Diego,Calif.). Briefly, Raw Blue™ cells that express human Toll Like Receptors(TLRs) (with the exception of TLR5) and an NF-κB/AP-1-inducible SEAP(secreted embryonic alkaline phosphatase) reporter gene were used.Stimulation of these cells with TR6 lead to NE-κB activation throughTLR1/2 which is measured by detection of SEAP levels. RAW-Blue™ Cellswere incubated with TR6 and LNP-TR6 at different concentration. Aftertwenty-four (24) hr. incubation with the compounds, TLR stimulation wasassessed by measuring the levels of SEAP optimal density (OD) using aQUANTI-Blue™ assay. ODs were normalized to the control (untreated)group. The result showed that treating the cells with TR6 and LNP-TR6could cause stimulation of TLR1/2 confirming the mechanism of action ofTR6 and its activity in LNP form. (See, FIG. 25 ).

Example 18: In Vitro Validation of TR8 Prodrug in Liposome formMechanism of Action

In another experiment, in vitro biological activity of LNP-TR8 and tofurther confirm targeting of TLR4, the following experiment(s) wereperformed using a RAW-Blue™ Cells and QUANTI-Blue™ assay (InvivoGen, SanDiego, Calif.). Briefly, Raw Blue™ cells that express human Toll LikeReceptors (TLRs) (with the exception of TLR5) and anNF-κB/AP-1-inducible SEAR (secreted embryonic alkaline phosphatase)reporter gene were used. Stimulation of these cells with TR8 lead toNF-κB activation through TLR4 which is measured by detection of SEAPlevels. RAW-Blue™ Cells were incubated with TR8 and LNP-TR8 at differentconcentration. After twenty-four (24) hr. incubation with the compounds,TLR stimulation was assessed by measuring the levels of SEAP optimaldensity (OD) using a QUANTI-Blue™ assay. ODs were normalized to thecontrol (untreated) group. The result showed that treating the cellswith TR8 and LNP-TR8 could cause stimulation of TLR4 confirming themechanism of action of TR8 and its activity in LNP form. (See, FIG. 26).

Example 19: In Vitro Validation of TR5 Prodrug in Liposome formMechanism of Action

In another experiment, in vitro biological activity of LNP-TR5 and tofurther confirm targeting of TLR7/8, the following experiment(s) wereperformed using a RAW-Blue™ Cells and QUANTI-Blue™ assay (InvivoGen, SanDiego, Calif.). Briefly, Raw Blue™ cells that express human Toll LikeReceptors (TLRs) (with the exception of TLR5) and anNF-κB/AP-1-inducible SEAP (secreted embryonic alkaline phosphatase)reporter gene were used. Stimulation of these cells with TR5 lead toNE-κB activation through TLR7/8 which is measured by detection of SEAPlevels. RAW-Blue™ Cells were incubated with TR5 and LNP-TR5 at differentconcentration. After twenty-four (24) hr. incubation with the compounds,TLR stimulation was assessed by measuring the levels of SEAP optimaldensity (OD) using a QUANTI-Blue™ assay. ODs were normalized to thecontrol (untreated) group. The result showed that treating the cellswith TR5 and LNP-TR5 could cause stimulation of TLR7/8 confirming themechanism of action of TR5 and its activity in LNP form. (See, FIG. 27).

Example 20: In Vitro Validation of TR3 Prodrug in Liposome FormMechanism of Action

In another experiment, in vitro biological activity of LNP-TR3 and tofurther confirm targeting of TLR7 and not targeting TLR8, the followingexperiment(s) were performed using a RAW-Blue™ Cells and QUANTI-Blue™,HEK-Blue™, and hTLR8 cell line assay (InvivoGen, San Diego, Calif.).Briefly, despite Raw Blue™ cells that express a variety of human TollLike Receptors (TLRs) (including TLR7/8 but with the exception of TLR5)hTLR8 cells dominantly express the human TLR8. Additionally, anNF-κB/AP-1-inducible SEAP (secreted embryonic alkaline phosphatase)reporter gene were used. Stimulation of these cells with TR3 lead toNF-κB activation through TLR8 which is measured by detection of SEAPlevels.

RAW-Blue™ Cells were incubated with TR5, LNP-TR5, TR3, TR3+Stearic Acid,and LNP-TR3 at different concentration. After twenty-four (24) hr.incubation with the compounds, TLR stimulation was assessed by measuringthe levels of SEAP optimal density (OD) using a QUANTI-Blue™ assay. ODswere normalized to the control (untreated) group. The results show bothTR5 and TR3 in prodrug and liposome form stimulated TLRs in RAW-Blue™cells. (See, FIG. 28 ).

Furthermore, HEK-Blue™ TLR8 Cells were incubated with TR5, LNP-TR5, TR3,TR3+Stearic Acid, and LNP-TR3 at different concentration. Aftertwenty-four (24) hr. incubation with the compounds, TLR stimulation wasassessed by measuring the levels of SEAP optimal density (OD) using aQUANTI-Blue™ assay. ODs were normalized to the control (untreated)group. The results show that only TR5 in prodrug and liposome form wereable to stimulate the cells. (See, FIG. 29 ).

Taken together, these results show that since TR5 is a TLR7/8 agonist,TR5 and TR5 in liposome form stimulate TLR7 and TLR8. However, TR3 canonly stimulate TLR7 and not TLR8. Thus, it is shown that TR3 is specificto TLR7 and not TLR8.

Example 21: Human Clinical Trials for the Treatment of Human CarcinomasThrough the Use of Formulated and/or Co-Formulated Liposomes ComprisingTLR Prodrugs

Formulated and/or co-formulated liposomes containing TLR prodrugs and/orTLR lipid moieties are used in accordance with the present inventionwhich specifically accumulate in a tumor cell and are used in thetreatment of certain tumors and other immunological disorders and/orother diseases. In connection with each of these indications, twoclinical approaches are successfully pursued.

I.) Adjunctive therapy: In adjunctive therapy, patients are treated withformulated and/or co-formulated liposomes containing TLR prodrugs incombination with a chemotherapeutic or pharmaceutical orbiopharmaceutical agent or a combination thereof. Primary cancer targetsare treated under standard protocols by the addition of formulatedand/or co-formulated liposomes containing TLR prodrugs. Protocol designsaddress effectiveness as assessed by the following examples, includingbut not limited to, reduction in tumor mass of primary or metastaticlesions, increased progression free survival, overall survival,improvement of patients health, disease stabilization, as well as theability to reduce usual doses of standard chemotherapy and otherbiologic agents. These dosage reductions allow additional and/orprolonged therapy by reducing dose-related toxicity of thechemotherapeutic or biologic agent.

II.) Monotherapy: In connection with the use of the formulated and/orco-formulated liposomes containing TLR prodrugs in monotherapy oftumors, the formulated and/or co-formulated liposomes containing TLRprodrugs are administered to patients without a chemotherapeutic orpharmaceutical or biological agent. In one embodiment, monotherapy isconducted clinically in end-stage cancer patients with extensivemetastatic disease. Protocol designs address effectiveness as assessedby the following examples, including but not limited to, reduction intumor mass of primary or metastatic lesions, increased progression freesurvival, overall survival, improvement of patients health, diseasestabilization, as well as the ability to reduce usual doses of standardchemotherapy and other biologic agents.

Dosage

Dosage regimens may be adjusted to provide the optimum desired response.For example, a single formulated and/or co-formulated liposomecontaining TLR prodrugs may be administered, several divided doses maybe administered over time or the dose may be proportionally reduced orincreased as indicated by the exigencies of the therapeutic situation.“Dosage Unit Form” as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention is dictated by and directly dependent on (a) theunique characteristics of the formulated and/or co-formulated liposomescontaining TLR prodrugs, (b) the individual mechanics of the combinationcompound, if any, (c) the particular therapeutic or prophylactic effectto be achieved, and (d) the limitations inherent in the art ofcompounding such a compound for the treatment of sensitivity inindividuals.

Clinical Development Plan (CDP)

The CDP follows and develops treatments of using formulated and/orco-formulated liposomes containing TLR prodrugs in connection withadjunctive therapy or monotherapy. Trials initially demonstrate safetyand thereafter confirm efficacy in repeat doses. Trials are open labelcomparing standard chemotherapy and/or the current standard of therapyplus formulated and/or co-formulated liposomes containing TLR prodrugs.As will be appreciated, one non-limiting criteria that can be utilizedin connection with enrollment of patients is expression of TLR in atumor as determined by standard detection methods known in the art.

It is believed that formulated and/or co-formulated liposomes, or any ofthe embodiments disclosed herein, may possess satisfactorypharmacological profile and promising biopharmaceutical properties, suchas toxicological profile, metabolism and pharmacokinetic properties,solubility, and permeability. It will be understood that determinationof appropriate biopharmaceutical properties is within the knowledge of aperson skilled in the art, e.g., determination of cytotoxicity in cellsor inhibition of certain targets or channels to determine potentialtoxicity.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models, methods, and life cycle methodology of the invention, inaddition to those described herein, will become apparent to thoseskilled in the art from the foregoing description and teachings, and aresimilarly intended to fall within the scope of the invention. Suchmodifications or other embodiments can be practiced without departingfrom the true scope and spirit of the invention.

TABLE I Examples of Lipids. No. Abbreviation Name/Chemical Formula 1CHOL Cholesterol 2 DPPG•Na1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) 3DMPG•Na 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodiumsalt) 4 Lyso PC 1-decanoy1-2-hydroxy-sn-glycero-3-phosphocholine 5(Δ9-Cis) PG 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodiumsalt) 6 Soy Lyso PC L-α-lysophosphatidylcholine (Soy) 7 PG1,2-dilauroyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) 8PA-PEG3-mannose 1,2-dipalmitoyl-sn-glycero-3-phospho((ethyl-1′,2′,3′-triazole)triethyleneglycolmannose) (ammonium salt) 9 C16 PEG2000N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene Ceramideglycol)2000]} 10  MPLA Monophosphoryl Lipid A

TABLE II Examples of Helper Lipids. No. Abbreviation Name 1 DOTAP1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) 2 DODMA1,2-dioleyloxy-3-dimethylaminopropane 3 DLinDMA1,2-dilinoleyloxy-3-dimethylaminopropane 4 DLin-KC2-DMA2,2-dilinoley1-4-dimethylaminoethyl-[1,3]-dioxolane 5 Δ9-Cis PE (DOPE)1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine 6 DOPC1,2-dioleoyl-sn-glycero-3-phosphocholine 7 CHOL Cholesterol 8 PEG-C-DMAN-[(methoxy poly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxlpropyl-3-amine 9 CHEMS cholesteryl hemisuccinate 10  DPPC1,2-dipalmitoyl-sn-glycero-3-phosphocholine 11  DSPC1,2-distearoyl-sn-glycero-3-phosphocholine 12  MO-CHOL4-(2-aminoethyl)-morpholino-cholesterolhemisuccinate

TABLE III Examples of Phospholipids/Fatty Acids. No. Name 1 Oleic acid 2linolenic acid 3 arachidonic acid 4 docosahexaenoic (DHA) 5 Palmiticacid 6 Palmitoleic acid 7 Stearic acid 8 Eicosapentaenoic acid (EPA) 9DSPE-PEG(2000) Carboxylic Acid (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol) 10  DOPE-PEG(2000)Carboxylic acid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (sodium salt)

The invention claimed is:
 1. A Toll-like receptor (TLR) prodrugcomposition comprising TR3 as the TLR prodrug, TR3 having the followingstructure:


2. A nanocarrier comprising the TLR prodrug of claim 1 whereby thenanocarrier releases an active TLR inhibitor after cleavage of thelinker unit joining the lipid moiety to the drug moiety.
 3. Thenanocarrier of claim 2, whereby the nanocarrier is further co-formulatedwith an iNKT (i-Natural Killer T cell) activator.
 4. The nanocarrier ofclaim 3, wherein the iNKT activator is Alpha-galactosylceramide(α-GalCer).
 5. The nanocarrier of claim 2, whereby the nanocarrier isfurther co-formulated with an immune modulating agent, wherein theimmune modulating agent is selected from the group consisting of otherTLR agonists and/or prodrugs, immunogenic-cell death inducingchemotherapeutics, IDO (indoleamine 2,3-dioxygenase) antagonists, STING(stimulator of interferon genes protein) agonists, CTLA-4 (cytotoxicT-lymphocyte-associated antigen 4) and inhibitors, PD-1/PD-L1(programmed cell death 1/programmed cell death ligand 1) inhibitorsand/or prodrugs thereof.
 6. The nanocarrier of claim 2, whereby thenanocarrier is further co-formulated with an ICD (immunogenic celldeath)-inducing chemotherapeutic, wherein the ICD-inducingchemotherapeutic is selected from the group consisting of Doxorubicin(DOX), Mitoxantrone (MTO), Oxaliplatin (OXA), Cyclophosphamide (CP),Bortezomib, Carfilzimib, or Paclitaxel.
 7. The nanocarrier of claim 2,further comprising doxorubicin (DOX).
 8. The nanocarrier of claim 2,wherein the nanocarrier comprises a liposome.
 9. The nanocarrier ofclaim 2, wherein the nanocarrier comprises a solid-lipid nanoparticle(SLNP).